Specific active site inhibitors of enzymes and methods of producing same

ABSTRACT

The present disclosure provides a method of producing enzyme-specific inhibitors or substrate binding partners comprising: identifying active site residues of the substrate in the enzyme substrate complex or in substrate binding partner-substrate complex; randomizing the active site residues to produce a combinatorial library of substrate variants; and selecting substrate variants that inhibit enzyme activity or bind substrate as substrate-specific binding partners. The present disclosure also provides ubiquitin enzyme specific inhibitors and ubiquitin variants that bind ubiquitin interaction motifs.

FIELD OF THE DISCLOSURE

The disclosure relates to specific active site inhibitors of substratebinding partners including, but not limited to, enzymes, and methods ofproducing said inhibitors. The disclosure also relates to ubiquitinvariants that specifically bind ubiquitin binding partners and, in someembodiments, inhibit enzymes.

BACKGROUND OF THE DISCLOSURE

Ubiquitination is a reversible post-translational modification thatplays major roles in multiple signaling events and in determining thelongevity of proteins in cells [Hershko and Ciechanover 1998; Glickmanand Ciechanover 2002; Chen 2005; Ye and Rape 2009]. Over the pastdecade, it has become clear that the biological importance ofubiquitination rivals and may exceed that of phosphorylation, andconsequently, there is great interest in deciphering the details of thisprocess in both normal and diseased cells.

The process of ubiquitination is hierarchical and involves an enzymecascade with increasing complexity [Hershko and Ciechanover 1998]. Inthe last step of the cascade, E3 ligases facilitate the transfer ofubiquitin (Ub) onto protein substrates through a covalent linkagebetween the C-terminal glycine of Ub and the ε-amino group of asubstrate lysine. Subsequently, polymeric Ub chains are extended on thesubstrate through linkages between the C termini and lysines ofadditional Ub monomers. The nature of these Ub-substrate and Ub-Ublinkages is precisely controlled by diverse Ub ligases, and in humans,more than 600 E3 ligases mediate substrate specificity.

Deubiquitinating enzymes (DUBs) counteract the processes initiated byubiquitination, and thus regulate cellular homeostasis and signaling.The human genome encodes approximately 95 putative DUBs which have beendivided into five structural families, as follows [Nijman, Luna-Vargaset al. 2005]: Ubiquitin specific proteases (USPs), Ubiquitin C-terminalhydrolases (UCHs), ovarian tumor proteases (OTUs), Machado-Josephdisease proteases (MJDs) and JAB1/MPN/MOV34 metalloenzymes (JAMMs).Aside from approximately eight JAMM family members, which aremetalloproteases, most known human DUBs are cysteine proteases. Thegeneral understanding of deubiquitination has lagged behind the generalunderstanding of ubiquitination within the scientific community, butseveral recent studies have revealed central roles for DUBs in thecontrol of cell signaling.

In particular, the largest DUB family consists of 58 USPs that areessential for many cellular processes and pathways. For example, USP21and USP22 have been implicated in the deubiquitination of histones[Nakagawa, Kajitani et al. 2008; Zhang, Varthi et al. 2008], while USP1,USP2a, USP7 and USP28 are involved in the DNA damage response [Cumminsand Vogelstein 2004; Li, Brooks et al. 2004; Nijman, Huang et al. 2005;Zhang, Zaugg et al. 2006; Stevenson, Sparks et al. 2007]. The USP familymembers share a structurally conserved catalytic domain with awell-defined catalytic cleft, suggesting that it may be possible todevelop a general structure-based strategy for inhibiting family membersby using similar yet specific molecular entities, as has been the casefor kinases that have been targeted with small molecules built on commonscaffolds [Fedorov, Marsden et al. 2007]. Unfortunately, no specificinhibitors of USPs or other DUBs have yet been reported, and a lack ofsuch inhibitors imposes a formidable roadblock to attempts to understandand manipulate deubiquitination pathways for therapeutic benefit.

Numerous USPs have been implicated in diseases includingneurodegeneration, haematological diseases, viral and bacterialinfections, and cancer [Goldenberg, McDermott et al. 2008]. Indeed, DUBsare direct antagonists of oncogenic and tumor-suppressive E3 ligases,and USPs are increasingly seen as potential targets for cancer therapy.Several USPs are up-regulated in cancer (e.g. USP2a, USP4, USP10) [Gray,Inazawa et al. 1995; Grunda, Nabors et al. 2006; Priolo, Tang et al.2006], are directly involved in the regulation of tumor-suppressiveproteins (e.g. USP2a and USP7) [Cummins and Vogelstein 2004; Li, Brookset al. 2004; Priolo, Tang et al. 2006] or carry mutations which arefound in hereditary cancers (CYLD) [Saggar, Chernoff et al. 2008]. USP8is implicated in ubiquitin remodeling, down regulation of epidermalgrowth factor receptor (EGFR), clathrin-mediated internalization,endosomal sorting, the control of receptor tyrosine kinases and it maybe involved in the patho-physiology of breast cancer [Mizuno, Iura etal. 2005; Avvakumov, Walker et al. 2006; Niendorf, Oksche et al. 2007].USP21 deubiquitinates histone 2A, and in doing so, influences themethylation status of histone 3, which has a major impact ontranscriptional control. More recently USP21 was also shown to beinvolved in NF-κB activation induced by tumor-necrosis factor α [Xu, Tanet al. 2010] and therefore could be involved in many disease areas suchas cancer, inflammation, viral infections and auto-immune diseases.

One of the best-studied examples of USP function is the role of USP7 inthe regulation of the tumor suppressor p53 and its associated E3 ligasemdm2. USP7 deubiquitinates both p53 and mdm2 but the net effect of itsfunction is to stabilize mdm2, and consequently, to destabilize p53.Thus, an inhibitor of USP7 would stabilize p53 and could be a potentialcancer therapeutic, because p53-induced apoptosis in response to DNAdamage has been proposed as a therapeutic strategy for several cancers[Chen 2005; Colland, Formstecher et al. 2009]. Mdm2 is alsodeubiquitinated by USP2a, which is up-regulated in prostate cancer[Priolo, Tang et al. 2006], and thus, inhibitors of USP2a would also bepromising therapeutics. Recently, it has been shown that USP10counteracts the effects of USP7 and USP2a by deubiquitinating andstabilizing p53 [Yuan, Luo et al. 2010].

USPs are multi-domain proteins that, in addition to a catalytic domain,typically contain various Ub recognition motifs and otherprotein-protein interaction domains [Komander, Clague et al. 2009].Although catalytic domains of different USPs often share low sequencehomology, crystal structures have revealed a common fold that definesthe family [Hu, Li et al. 2002; Reyes-Turcu, Ventii et al. 2009] and acommon catalytic triad that mediates catalysis [Wilkinson 1997]. The pKaof the catalytic cysteine is lowered by a histidine, and a thirdresidue, usually asparagine or aspartate, polarizes and aligns thehistidine side-chain.

Structures of five USP catalytic domains in complex with Ub also reveala common binding site for the substrate [Hu, Li et al. 2002; Hu, Li etal. 2005; Renatus, Parrado et al. 2006]. In all cases, Ub is bound inthe same orientation and the isopeptide linkage is aligned in the activesite. While the affinity of USPs for Ub is low, the contact surfacebetween Ub and the USP is large, as for example, the contact surface ofthe Ub and USP7 complex is known to be 1800 Å². Notably, despite acommon function, the Ub-binding sites of USP family members differ insequence, and consequently, the Ub-binding surfaces are similar butexhibit significant topological variation. In the case of USP7,approximately 75% of the Ub-binding surface is composed of residues thatare not conserved in the USP family.

In US 2006/0099686 A1, a modified Ub was used to establish analternative binding-scaffold to a predetermined binding partner it didnot recognize before. The modified ubiquitin had the point mutationsI44A, K48R, R54L, V70A, R72L, G75A and the last glycine in the proteinwas removed. These mutations prevented ubiquitin from interacting withits natural binding partner and avoided conjugation with other ubiquitinmolecules through Lys48. In this modified ubiquitin the inventors alsorandomized the residues 2, 4 and 6 in the N-terminal part and residues62-66 in addition to the point mutations to produce a continuous surfaceon one side of the ubiquitin and used phage display to select for highaffinity variants to hydrocortisone (hapten) and proteins such asvascular endothelial growth factor (VEGF) and Fc part of IgG antibodies.They achieved affinities in the 170 nM-10 μM range. The surface ofubiquitin is not particularly well suited to generate binding surfacesto haptens since it lacks a cavity to allow an efficient shielding ofthe hydrophobic surface of a molecule like hydrocortisone. In addition,the solvent accessible binding surface covered by these residues isrelatively small (500 Å²), and does not provide enough structuraldiversity for an efficient binding of other proteins. This explains thecomparable low affinity interactions the inventors have observed whichmakes a diagnostic or pharmaceutical usage difficult.

SUMMARY OF THE DISCLOSURE

The present disclosure focuses on the existing interactions ofsubstrates with their binding partners, including enzymes, and using asan example, the ubiquitination pathway that already have a ubiquitin(Ub)-binding site, to develop higher affinity binding partners that aremore suited to diagnostic or pharmaceutical usage. The presentdisclosure provides a method of preparing ubiquitin variants thatspecifically bind to a ubiquitin specific protease (USP), a ubiquitinligase, or a ubiquitin interaction motif (UIM). Based on this method,specific Ub variants were identified that bind tightly to particularUSPs, ubiquitin ligases, and UIMs, but not to others. These specific Ubvariants act as potent, competitive inhibitors of USP activity byblocking the binding or interfering with the binding of Ub substrates totheir binding partners, i.e. USP, ubiquitin ligase, or UIM.

The disclosure includes a ubiquitin binding partner, wherein theubiquitin binding partner is a ubiquitin polypeptide or a fragmentthereof comprising an amino acid mutation in a region selected from thegroup consisting of: (a) region 1 (amino acids 2-14), region 2 (aminoacids 35-49), or region 3 (amino acids 62-72) of the amino acid sequenceof ubiquitin (Ub) set forth in SEQ ID NO:1; and (b) region 1 (aminoacids 2-14), region 2 (amino acids 42-49), or region 3 (amino acids62-78) of the amino acid sequence of a ubiquitin variant (Ubv) set forthin SEQ ID NO:21. In some aspects, the ubiquitin binding partner is aubiquitin-interacting motif (UIM) or a ubiquitin enzyme inhibitor. Insome aspects, the ubiquitin binding partner inhibits a ubiquitin enzymeselected from the group consisting of: a ubiquitin-activating enzyme (E1enzyme), a ubiquitin-conjugating enzyme (E2 enzyme), a ubiquitin ligase(E3 enzyme), and a deubiquitinating enzyme. In some aspects, theubiquitin binding partner is a ubiquitin ligase. In some aspects, theubiquitin ligase is neural precursor cell expressed developmentallydown-regulated protein 4 (Nedd4) or ITCH. In some aspects, the ubiquitinbinding partner is a deubiquitinating enzyme. In some aspects, thedeubiquitinating enzyme is a ubiquitin-specific protease (USP), aubiquitin C-terminal hydrolase (UCH), an ovarian tumor-related protease(OTU), a Machado-Joseph disease (MJD) protease or a JAB1/MPN/MOV34metalloenzyme (JAMM) protease. In other aspects, the deubiquitinatingenzyme is a ubiquitin-specific protease (USP).

The disclosure includes a ubiquitin binding partner wherein theubiquitin binding partner is a ubiquitin polypeptide or a fragmentthereof comprising an amino acid mutation wherein the mutation is asubstitution in a region of a ubiquitin polypeptide or a fragmentthereof selected from the group consisting of: (a) region 1 (amino acids2-14) wherein the polypeptide comprises the structure:

(SEQ ID NO: 173) X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄,wherein,X₂ is selected from the group consisting of Arg, Tyr, Asp, His, Glu,Pro, Leu, Thr and Lys;X₄ is selected from the group consisting of Val, Asp, Met, Tyr, Ser, Ileand Leu;X₆ is selected from the group consisting of Asn, Met, Ile, Gln, His,Arg, Glu, Thr, Pro, Val, Gly, Leu and Tyr;X₈ is selected from the group consisting of Phe, Gly, Ile, Arg, Pro,Met, His, Val and Ser;X₉ is selected from the group consisting of Met, Trp, Ser, Ala, Leu,Val, Ile, Tyr, Asn, Arg, Lys and Pro;X₁₀ is selected from the group consisting of Thr, Ala, Leu, Met, Arg,Gln, Trp, Val, His and Glu;X₁₁ is selected from the group consisting of Arg, Thr, Asn, Glu, Trp,Phe, Asp, Met, Tyr, Gly, Gln, His, Leu, Ser and Ala;X₁₂ is selected from the group consisting of Ile, His, Ala, Asn, Ser,Met, Val, Gly, Arg, Asp and Phe;X₁₄ is selected from the group consisting of Ile, Tyr, Asn, Arg, Phe,Pro, Gln, Ala, Met, His, Ser, Gly, Asp, Glu and Leu;(b) region 2 (amino acids 35-49) wherein the polypeptide comprises thestructure:

(SEQ ID NO: 173) X₃₅-X₃₆-X₃₇-X₃₈-X₃₉-X₄₀-X₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆- X₄₇,wherein,X₃₅ is Gln;X₃₇ is Asp;X₄₂ is selected from the group consisting of Thr, Ser, Lys, Phe, Ile,Tyr, Asn, and Gly;X₄₄ is selected from the group consisting of Val, Phe, Thr, Asn, Leu,Ser, and Tyr;X₄₆ is selected from the group consisting of Asp, Val, Thr, Asn, Phe,Gly, Ser and Ile;X₄₇ is selected from the group consisting of Thr, Arg, Trp, Phe, Arg,Lys, Ala, and Val;X₄₈ is selected from the group consisting of Gln, Asn, Thr, Met, Leu,Asp, Arg, and Trp;X₄₉ is selected from the group consisting of Arg, Leu, Pro, Lys, Thr,Ile, His, Phe, and Glu; and (c) region 3 (amino acids 62-78) wherein thepolypeptide comprises the structure:

(SEQ ID NO: 173) X₆₂-X₆₃-X₆₄-X₆₅-X₆₆-X₆₇-X₆₈-X₆₉-X₇₀-X₇₁-X₇₂-X₇₃-X₇₄-X₇₅-X₇₆-X₇₇-X₇₈,wherein,X₆₂ is selected from the group consisting of His, Ser, Leu, Pro, Arg,Gly, Lys, Glu, Asn, Tyr and Val;X₆₃ is selected from the group consisting of Asn, Arg, Lys, Pro, Thr,Met, Tyr, Gln, Gly, Trp, His and Leu;X₆₄ is selected from the group consisting of His, Val, Ile, Arg, Ser,Trp, Asp, Tyr, Lys, Gln, Phe, Gly, Ala, Thr, Leu and Asn;X₆₆ is selected from the group consisting of Ala and Tyr;X₆₈ is selected from the group consisting of Tyr, Phe, Ala, Arg, Pro,Gly, Lys, Leu, Ser, Gln, Asp, and Asn;X₇₀ is selected from the group consisting of Leu, Lys, Phe, Trp, Ala,Gly, Met, and Ile;X₇₁ is selected from the group consisting of Phe, Met, Val, Lys, Gly,Arg, Trp, Ser, Ala, Thr and Ile;X₇₂ is selected from the group consisting of Lys, Thr, Ile, Trp, Ser,Met and Gly;X₇₃ is selected from the group consisting of Met, Phe, Asp, Ile, Pro,Arg, His and Val;X₇₄ is selected from the group consisting of Trp, Leu, Tyr, His, Phe,Ser, Pro, Ile, Gly and Thr;X₇₅ is selected from the group consisting of Ser, Arg, Ala, Leu, Val,Thr, Asp, Trp and Phe;X₇₆ is selected from the group consisting of His, Glu, Val, Gln, Leu,Cys, Asn, Ala, Trp, Ile, Arg, Tyr, Met, Thr Asp and Lys;X₇₇ is selected from the group consisting of Arg, Lys, Ala, Ile, Ser,Asp, Asn, Phe, Glu, Trp, Gln, His, Leu, Val, Tyr, Thr and Met; andX₇₈ is selected from the group consisting of Arg, Thr, Ala, Gln, His,Lys, Met, Val, Leu, Ile, Phe, Cys, Glu, Tyr, Ser, Asn and Pro.

In some aspects, the ubiquitin binding partner comprises an amino acidmutation at position 2, 4, 6, 8, 9, 10, 11, 12, 14, 35, 37, 39, 40, 42,44, 46, 47, 48, 49, 62, 63, 64, 66, 68, 70, 71 or 72 in the amino acidsequence of ubiquitin set forth in SEQ ID NO:1. In other aspects, theubiquitin binding partner comprises an amino acid mutation at position2, 4, 6, 8, 9, 10, 11, 12, 14, 62, 63, 64, 66, 68, 70, 71 or 72 in theamino acid sequence of ubiquitin set forth in SEQ ID NO: 1. In moreparticular aspects, the ubiquitin binding partner comprises an aminoacid mutation at position 2, 4, 8, 9, 11, 14, 62, 63, 64, 66, 68, 70, 71or 72 in the amino acid sequence of ubiquitin set forth in SEQ ID NO:1.In some aspects, the ubiquitin binding partner binds toubiquitin-specific protease 8 (USP8). In some aspects, the ubiquitinbinding partner comprises the amino acid sequence of any one of SEQ IDNOS: 2-3.

In some aspects, the ubiquitin binding partner comprises an amino acidmutation at position 8, 14, 62, 64, 68, 70, 71 or 72 in the amino acidsequence of ubiquitin set forth in SEQ ID NO: 1. In some aspects, theubiquitin binding partner binds to ubiquitin-specific protease 21(USP21). In some aspects, the ubiquitin binding partner comprises anamino acid sequence of any one of SEQ ID NOS: 4-8.

In some aspects, the ubiquitin binding partner comprises an amino acidmutation at position 2, 4, 6, 8, 9, 10, 11, 12, 14, 42, 44, 46, 47, 48,49, 62, 63, 64, 68, 70, 71, 72, 73, 74, 75, 76, 77, or 78 in the aminoacid sequence of a ubiquitin variant (Ubv) set forth in SEQ ID NO: 21.In other aspects, the ubiquitin binding partner comprises an amino acidmutation at position 2, 6, 8, 9, 10, 11, 12, 62, 63, 64, 68, 70, 71, 74,75, 76, 77, or 78 in the amino acid sequence of the ubiquitin variant(Ubv) set forth in SEQ ID NO: 21. In some aspects, the ubiquitin bindingpartner binds to ubiquitin-specific protease 2a (USP2a). In someaspects, the ubiquitin binding partner comprises an amino acid sequenceof any one of SEQ ID NOS: 22-47. In particular aspects, the ubiquitinbinding partner binds to USP2a and increases cell death. In someaspects, the cell death is apoptosis. In some aspects, the cells arecancer cells. In certain aspects, the cancer cells are prostate cancercells.

In some aspects, the ubiquitin binding partner comprises an amino acidmutation at position 2, 4, 6, 8, 9, 10, 11, 12, 14, 42, 46, 47, 48, 49,62, 63, 64, 68, 70, 71, 73, 74, 75, 76, 77, or 78 in the amino acidsequence of the ubiquitin variant (Ubv) set forth in SEQ ID NO:21. Insome aspects, the ubiquitin binding partner binds to ubiquitin-specificprotease USP5. In some aspects, the ubiquitin binding partner comprisesthe amino acid sequence set forth in any one of SEQ ID NOS: 48-68. Inparticular aspects, the ubiquitin binding partner increasestranscription of tumor suppressor protein p53 and or increases freepoly-ubiquitin. In some aspects, increases in transcription of p53decreases proliferation of p53-dependent tumor cells. In certainaspects, the tumor cells are cancer cells.

In some aspects, the ubiquitin binding partner comprises an amino acidmutation at position 6, 8, 9, 14, 44, 46, 49, 63, 64, 68, 70, 71, 74,75, 76, 77, or 78 in the amino acid sequence of the ubiquitin variant(Ubv) set forth in SEQ ID NO: 21. In some aspects, the ubiquitin bindingpartner binds ubiquitin-specific protease 10 (USP10). In some aspects,the ubiquitin binding partner comprises the amino acid sequence setforth in SEQ ID NO: 69. In some aspects, the ubiquitin binding partnerincreases transcription of tumor suppressor protein p53. In particularaspects, such increase in transcription of p53 decreases proliferationof p53-dependent tumor cells. In some aspects, such tumor cells arecancer cells.

In some aspects, the ubiquitin binding partner comprises an amino acidmutation at position 2, 4, 6, 8, 9, 11, 12, 14, 44, 46, 47, 48, 49, 62,63, 64, 68, 70, 73, 74, 76, 77, or 78 in the amino acid sequence of theubiquitin variant (Ubv) set forth in SEQ ID NO:21. In some aspects, theubiquitin binding partner binds ubiquitin-specific protease 48 (USP48).In some aspects, the ubiquitin binding partner comprises the amino acidsequence set forth in any one of SEQ ID NOS: 70-74. In particularaspects, the ubiquitin binding partner decreases proliferation of cancercells. In some aspects, such cancer cells are pancreatic cancer cells.

In some aspects, the ubiquitin binding partner comprises an amino acidmutation at position 2, 4, 6, 8, 9, 10, 11, 12, 14, 42, 44, 46, 47, 48,49, 62, 63, 64, 68, 70, 71, 72, 73, 74, 75, 76, 77, or 78 in the aminoacid sequence of the ubiquitin variant (Ubv) set forth in SEQ ID NO:21.In some aspects, such ubiquitin binding partner binds neural precursorcell expressed developmentally down-regulated protein 4 (Nedd4). In someaspects, such ubiquitin binding partner comprises the amino acidsequence set forth in any one of SEQ ID NOS: 75-98. In some aspects, theubiquitin binding partner binds E3 ubiquitin ligase ITCH. In someaspects, the ubiquitin binding partner comprises the amino acid sequenceset forth in any one of SEQ ID NOS: 99-104. In some aspects, suchbinding to ITCH results in decreased ubiquitination of proteins. In someaspects, such binding to ITCH decreases proliferation of tumor cells. Infurther aspects, the ubiquitin binding partner binds ubiquitininteraction motifs (UIM). In some aspects, the ubiquitin binding partnercomprises the amino acid sequence set forth in any one of SEQ ID NOS:105-129.

In some aspects, the ubiquitin binding partner comprises an amino acidmutation at position 6, 8, 9, 11, 12, 14, 42, 44, 46, 47, 49, 62, 63,64, 68, 70, 71, 72, 73, 74, 75, 76, 77, or 78 in the amino acid sequenceof the ubiquitin variant (Ubv) set forth in SEQ ID NO:21. In someaspects, the ubiquitin binding partner binds to ubiquitin-specificprotease 8 (USP8). In some aspects, the ubiquitin binding partnercomprises the amino acid sequence set forth in any one of SEQ ID NOS:130-140.

In some aspects, the ubiquitin binding partner comprises an amino acidmutation at position 2, 6, 8, 9, 10, 11, 12, 14, 62, 63, 64, 68, 70, 71,72, 73, 74, 75, 76, 77, or 78 in the amino acid sequence of theubiquitin variant (Ubv) set forth in SEQ ID NO: 21. In some aspects, theubiquitin binding partner binds to ubiquitin-specific protease 21(USP21). In some aspects, the ubiquitin binding partner comprises theamino acid sequence set forth in any one of SEQ ID NOS: 141-169.

The disclosure also includes nucleic acids encoding any of the ubiquitinbinding partners described herein. The disclosure further includesrecombinant expression vectors comprising such nucleic acid molecules.The disclosure also includes host cells comprising such nucleic acidmolecules or recombinant expression vectors.

The disclosure also includes methods of identifying a substrate variantas a substrate-specific binding partner comprising: (a) randomizingactive site residues of a substrate in an enzyme substrate complex or ina complex comprising a substrate binding partner and a substrate toproduce a combinatorial library of substrate variants; and (b) selectingthe substrate variant that binds the substrate as a substrate-specificbinding partner. In some aspects, such active site residues areidentified by a method selected from analysis of 3D structures, alaninescanning, shotgun scanning or mutational analysis of interactioninterfaces. In some aspects, such randomizing of the active siteresidues is performed by genetic engineering. In certain aspects, therandomizing is performed to yield 75% wild type amino acid residues and25% mutated amino acid residues in the active site residues.

In some aspects, selecting the substrate variant that inhibits enzymeactivity or binds substrate is performed by phage display to determineinhibition of enzyme activity or substrate binding in the variantcompared to a control. In certain aspects, when the substrate bindingpartner is an enzyme, the control is an enzyme different than the enzymein the enzyme substrate complex thereby selecting for enzymespecificity. In other aspects, when the substrate binding partner is anenzyme the control is the enzyme in the enzyme substrate complex therebytesting for amount of enzyme activity inhibition. In some aspects, suchinhibition is competitive inhibition, product inhibition or allostericinhibition. In some aspects, the enzyme is a transferase, hydrolase,lyase, or ligase. In further aspects, the substrate is ubiquitin (Ub)and the enzyme is a ubiquitin-activating enzyme (E1 enzyme), aubiquitin-conjugating enzyme (E2 enzyme), a ubiquitin ligase (E3enzyme), and a deubiquitinating enzyme. In particular aspects, suchubiquitin ligase is neural precursor cell expressed developmentallydown-regulated protein 4 (Nedd4) or ITCH. In more particular aspects,such deubiquitinating enzyme is a ubiquitin specific protease (USP), aubiquitin C-terminal hydrolase (UCH), an ovarian tumor protease (OTU), aMachado-Joseph disease (MJD) protease or a JAB1/MPN/MOV34 metalloenzyme(JAMM) protease. In other aspects, the deubiquitinating enzyme is aubiquitin specific protease (USP). In some aspects, such USP is USP2,USP5, USP8, USP10, USP21 or USP48. In other aspects, such methods asdescribed herein above are included wherein the substrate is ubiquitin(Ub) and the substrate binding partner is a ubiquitin-interacting motif(UIM).

The disclosure also includes methods of identifying a substrate variantas a substrate-specific binding partner, wherein the substrate isubiquitin as set forth in SEQ ID NO:1 or a ubiquitin variant as setforth in SEQ ID NO: 21 and the ubiquitin binding partner is a ubiquitinpolypeptide or a fragment thereof comprising an amino acid mutation in aregion selected from the group consisting of: (a) region 1 (amino acids2-14), region 2 (amino acids 35-49), or region 3 (amino acids 62-72) ofthe amino acid sequence of ubiquitin (Ub) set forth in SEQ ID NO:1; and(b) region 1 (amino acids 2-14), region 2 (amino acids 42-49), or region3 (amino acids 62-78) of the amino acid sequence of a ubiquitin variant(Ubv) set forth in SEQ ID NO:21. In some aspects, such mutation is asubstitution in a region of a ubiquitin polypeptide or a fragmentthereof selected from the group consisting of:

(a) region 1 (amino acids 2-14) wherein the polypeptide comprises thestructure:

(SEQ ID NO: 173) X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄,wherein,X₂ is selected from the group consisting of Arg, Tyr, Asp, His, Glu,Pro, Leu, Thr and Lys;X₄ is selected from the group consisting of Val, Asp, Met, Tyr, Ser, Ileand Leu;X₆ is selected from the group consisting of Asn, Met, Ile, Gln, His,Arg, Glu, Thr, Pro, Val, Gly, Leu and Tyr;X₈ is selected from the group consisting of Phe, Gly, Ile, Arg, Pro,Met, His, Val and Ser;X₉ is selected from the group consisting of Met, Trp, Ser, Ala, Leu,Val, Ile, Tyr, Asn, Arg, Lys and Pro;X₁₀ is selected from the group consisting of Thr, Ala, Leu, Met, Arg,Gln, Trp, Val, His and Glu;X₁₁ is selected from the group consisting of Arg, Thr, Asn, Glu, Trp,Phe, Asp, Met, Tyr, Gly, Gln, His, Leu, Ser and Ala;X₁₂ is selected from the group consisting of Ile, His, Ala, Asn, Ser,Met, Val, Gly, Arg, Asp and Phe;X₁₄ is selected from the group consisting of Ile, Tyr, Asn, Arg, Phe,Pro, Gln, Ala, Met, His, Ser, Gly, Asp, Glu and Leu;(b) region 2 (amino acids 35-49) wherein the polypeptide comprises thestructure:

(SEQ ID NO: 173) X₃₅-X₃₆-X₃₇-X₃₈-X₃₉-X₄₀-X₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆- X₄₇,wherein,X₃₅ is Gln;X₃₇ is Asp;X₄₂ is selected from the group consisting of Thr, Ser, Lys, Phe, Ile,Tyr, Asn, and Gly;X₄₄ is selected from the group consisting of Val, Phe, Thr, Asn, Leu,Ser, and Tyr;X₄₆ is selected from the group consisting of Asp, Val, Thr, Asn, Phe,Gly, Ser and Ile;X₄₇ is selected from the group consisting of Thr, Arg, Trp, Phe, Arg,Lys, Ala, and Val;X₄₈ is selected from the group consisting of Gln, Asn, Thr, Met, Leu,Asp, Arg, and Trp;X₄₉ is selected from the group consisting of Arg, Leu, Pro, Lys, Thr,Ile, His, Phe, and Glu; and(c) region 3 (amino acids 62-78) wherein the polypeptide comprises thestructure:

(SEQ ID NO: 173) X₆₂-X₆₃-X₆₄-X₆₅-X₆₆-X₆₇-X₆₈-X₆₉-X₇₀-X₇₁-X₇₂-X₇₃-X₇₄-X₇₅-X₇₆-X₇₇-X₇₈,wherein,X₆₂ is selected from the group consisting of His, Ser, Leu, Pro, Arg,Gly, Lys, Glu, Asn, Tyr and Val;X₆₃ is selected from the group consisting of Asn, Arg, Lys, Pro, Thr,Met, Tyr, Gln, Gly, Trp, His and Leu;X₆₄ is selected from the group consisting of His, Val, Ile, Arg, Ser,Trp, Asp, Tyr, Lys, Gln, Phe, Gly, Ala, Thr, Leu and Asn;X₆₆ is selected from the group consisting of Ala and Tyr;X₆₈ is selected from the group consisting of Tyr, Phe, Ala, Arg, Pro,Gly, Lys, Leu, Ser, Gln, Asp, and Asn;X₇₀ is selected from the group consisting of Leu, Lys, Phe, Trp, Ala,Gly, Met, and Ile;X₇₁ is selected from the group consisting of Phe, Met, Val, Lys, Gly,Arg, Trp, Ser, Ala, Thr and Ile;X₇₂ is selected from the group consisting of Lys, Thr, Ile, Trp, Ser,Met and Gly;X₇₃ is selected from the group consisting of Met, Phe, Asp, Ile, Pro,Arg, His and Val;X₇₄ is selected from the group consisting of Trp, Leu, Tyr, His, Phe,Ser, Pro, Ile, Gly and Thr;X₇₅ is selected from the group consisting of Ser, Arg, Ala, Leu, Val,Thr, Asp, Trp and Phe;X₇₆ is selected from the group consisting of His, Glu, Val, Gln, Leu,Cys, Asn, Ala, Trp, Ile, Arg, Tyr, Met, Thr Asp and Lys;X₇₇ is selected from the group consisting of Arg, Lys, Ala, Ile, Ser,Asp, Asn, Phe, Glu, Trp, Gln, His, Leu, Val, Tyr, Thr and Met; andX₇₈ is selected from the group consisting of Arg, Thr, Ala, Gln, His,Lys, Met, Val, Leu, Ile, Phe, Cys, Glu, Tyr, Ser, Asn and Pro.In some aspects of the disclosed methods, the ubiquitin binding partnercomprises an amino acid mutation at position 2, 4, 6, 8, 9, 10, 11, 12,14, 35, 37, 39, 40, 42, 44, 46, 47, 48, 49, 62, 63, 64, 66, 68, 70, 71or 72 in the amino acid sequence of ubiquitin set forth in SEQ ID NO: 1.In some other aspects of the disclosed methods, the ubiquitin bindingpartner comprises an amino acid mutation at position 2, 4, 6, 8, 9, 10,11, 12, 14, 42, 44, 46, 47, 48, 49, 62, 63, 64, 68, 70, 71, 72, 73, 74,75, 76, 77, or 78 in the amino acid sequence of a ubiquitin variant setforth in SEQ ID NO: 21.

Accordingly, in more specific aspects, the present disclosure provides amethod of producing enzyme-specific inhibitors comprising: a)identifying active site residues of a substrate in an enzyme-substratecomplex; b) randomizing the active site residues to produce acombinatorial library of substrate variants; and c) selecting substratevariants that inhibit enzyme activity.

The present disclosure also includes uses of the ubiquitin variantsdescribed herein for methods of inducing cell death in various celltypes. In some aspects, the cell death is apoptosis. In some aspects,the ubiquitin variants described herein are used in the treatment ofcancer or in a medicament for the treatment of cancer.

In further aspects, the ubiquitin variants described herein are used inscreening small molecule libraries for inhibitors of the ubiquitinpathway, including inhibitors of USPs, OTUs, E3 ligases andE2-conjugating enzymes.

In more specific aspects, the active site residues of ubiquitin compriseregions 1-3 as shown in FIG. 1.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the disclosure aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will now be described in relation to the drawings inwhich:

FIG. 1 shows the Ub library design. (A) The library design mapped ontothe Ub structure (PDB entry 1 UBQ). The Ub main-chain is shown as ablack tube and positions that were diversified in the library are shownas spheres, highlighted as follows: region 1 (light gray), region 2(gray), region 3 (dark gray). (B) The primary sequence of the regionstargeted in the library design. Diversified sequences are shaded andhighlighted as in panel A.

FIG. 2 shows the sequences and specificity profiles of Ub variants. (A)Sequence alignment of Ub.wt (SEQ ID NO: 1) and Ub variants selected forbinding to USP8 or USP21. The alignment shows only those positions thatwere diversified in the Ub library, and positions that were conserved asthe wt sequence are indicated by dashes. (B) The Ub variants wereassayed by phage ELISA for binding to the following immobilized USPproteins: USP2 (downward diagonal), USP7 (upward diagonal), USP8(black), USP10 (grid), USP21 (gray). Bound phage were detectedspectrophotometrically (optical density at 450 nm) and backgroundbinding to neutravidin was subtracted from the signal.

FIG. 3 shows phage-derived Ub variants are potent and specificinhibitors of USPs. (A) The activity of USP8 (white bars) or USP21 (greybars) in the presence of 1.0 μM Ub.wt (SEQ ID NO: 1), Ubv.8.2 (SEQ IDNO: 2) or Ubv.21.3 (SEQ ID NO: 6). The activity of USP8 (1.0 nM) orUSP21 (25 nM) was measured with Ub-AMC substrate at 1.0 μM, and isnormalized to the activity in the absence of Ub variants. Dose responsecurves for the activity of USP21 (B) or USP8 (C) (right panel) in thepresence of varying concentrations of Ubv.8.2 (SEQ ID NO: 2) or UBv.21.3(SEQ ID NO: 6), respectively. The IC₅₀ value was determined as theconcentration of Ub variant that reduced USP activity by 50%. Theactivity of USP8 (1.0 nM) or USP21 (25 nM) was measured with Ub-AMCsubstrate at 1.0 μM, and is normalized to the activity in the absence ofUb variants.

FIG. 4 shows USPs in complex with corresponding inhibitors. Ub-variantsare shown in dark gray tubes. Changes contributing to an enhancedaffinity from region 1 are shown as light gray spheres and from region 3as dark gray spheres. (A) USP21 in complex with the Ubv.21.4 (SEQ ID NO:7) (B) USP8 in complex with Ubv.8.2 (SEQ ID NO: 2).

FIG. 5 shows phage-derived Ub variants are potent and specificinhibitors of USP2. (A) Sequence alignment of Ub.wt variant comprisingtwo additional C-terminal amino acids (two glycines at positions 76a and76b, i.e., amino acids 77 and 78 of SEQ ID NO: 21) with phage-derivedvariants that bind USP2. The alignment shows only those randomizedpositions that were divergent from Ub.wt (SEQ ID NO: 1), and positionsthat were conserved as the wt sequence are indicated by dashes. (B)ELISA demonstrating binding specificity of Ub variants to various USPs,UIMs, ITCH, and NEDD4. Selected phages were tested against Ub-bindingdomains.

FIG. 6 shows phage-derived Ub variants are potent and specificinhibitors of USP5. (A) Sequence alignment of Ub.wt variant comprisingtwo additional C-terminal amino acids (two glycines at positions 76a and76b, i.e., amino acids 77 and 78 of SEQ ID NO: 21) with phage-derivedvariants that bind USP5. The alignment shows only those randomizedpositions that were divergent from Ub.wt (SEQ ID NO: 1), and positionsthat were conserved as the wt sequence are indicated by dashes. (B)ELISA demonstrating binding specificity of selected Ubv, i.e. ubiquitinenzyme inhibitors, with binding activity to USP5. Phage showsintrinsically non-specific binding to USP2a, explaining the high bindingsignal of selected Ubvs with binding activity to USP5.

FIG. 7 shows the identification of one Ub variant (Ubv.10.1 (SEQ ID NO:69)) as a potent and specific inhibitor of USP10. (A) Sequence alignmentof Ub.wt variant (SEQ ID NO: 21) with variant that binds USP10. Thealignment shows only the 17 randomized positions that were divergentfrom Ub.wt (SEQ ID NO: 1). (B) ELISA demonstrating binding specificityof Ubv.10.1 (SEQ ID NO: 69) to USP10.

FIG. 8 shows the identification of five Ub variants (Ubv.48.01-Ubv.48.05(SEQ ID NOs: 70-74, respectively)) as potent and specific inhibitors ofUSP48. (A) Sequence alignment of Ub.wt variant (SEQ ID NO: 21) withvariants that bind USP48. The alignment shows only those randomizedpositions that were divergent from Ub.wt (SEQ ID NO: 1), and positionsthat were conserved as the wt sequence are indicated by dashes. (B)ELISA demonstrating binding specificity of Ubv to USP48.

FIG. 9 shows the identification of Ub variants as potent and specificinhibitors of Nedd4. (A) Sequence alignment of Ub.wt variant (SEQ ID NO:21) with variants that binds Nedd4. Positions that were conserved as thewt sequence are indicated by dashes (B) ELISA demonstrating bindingspecificity of Ubvs to Nedd4.

FIG. 10 shows the identification of Ub variants as potent and specificinhibitors of ITCH. (A) Sequence alignment of Ub.wt variant (SEQ ID NO:21) with variants that binds ITCH. Positions that were conserved as thewt sequence are indicated by dashes. (B) ELISA demonstrating bindingspecificity of Ubvs to ITCH.

FIG. 11 shows the identification of Ub variants as potent and specificinhibitors of UIM. (A) Sequence alignment of Ub.wt variant (SEQ ID NO:21) with variants that binds UIM. Positions that were conserved as thewt sequence are indicated by dashes. (B) ELISA demonstrating bindingspecificity of Ubvs to UIMs (peptides designated Rensselaer PolytechGeorge Makhadatze (RPGM)-1, RPGM-2, and RPGM-3, (UIM1, UIM1a, and UIM1b,respectively) from Rensselaer Polytechnic Institute).

FIG. 12 shows the identification of additional Ub variants as potent andspecific inhibitors of USP8. (A) Sequence alignment of Ub.wt variant(SEQ ID NO: 21) with variants that binds USP8. Positions that wereconserved as the wt sequence are indicated by dashes. (B) ELISAdemonstrating binding specificity of Ubvs to USP8.

FIG. 13 shows the identification of additional Ub variants as potent andspecific inhibitors of USP21. (A) Sequence alignment of Ub.wt variant(SEQ ID NO: 21) with variants that binds USP21. Positions that wereconserved as the wt sequence are indicated by dashes. (B) ELISAdemonstrating binding specificity of Ubvs to USP21.

FIG. 14 shows dose response curves for the inhibition of USP8 and USP21by Ubv.8.2 (SEQ ID NO: 2) (A) and Ubv.21.4 (SEQ ID NO: 7) (B),respectively.

FIG. 15 shows Ubv.21.4 (SEQ ID NO: 7) CA2 mutant specifically inhibitsUSP21 activity through intracellular interaction with USP21. (A) NF-κBand control Renilla luciferase reporter plasmids were cotransfected intoHEK293T cells with increased amounts of expression plasmid for USP21; 36hours later, transfected cells were treated with TNFα for 6 hours. NF-κBactivation in the cells was determined by measuring luciferase activityin the cell lysates. (B) NF-κB and control Renilla luciferase reporterplasmids were co-transfected into HEK293T cells with expression plasmidfor RIP1 as well as increased amounts of expression plasmid for USP21;36 hours later, NF-κB activation in the cells was determined bymeasuring luciferase activity in the cell lysates. (C) NF-κB and controlRenilla luciferase reporter plasmids were co-transfected into HEK293Tcells with expression plasmid for USP21 as well as increased amounts ofexpression plasmid for Ubv.21.4 (SEQ ID NO: 7) or Ub.wt (SEQ ID NO: 1);36 hours later, cells were treated with TNFα for 6 hours. NF-κBactivation in the cells was determined by measuring luciferase activityin the cell lysates. (D) NF-κB and control Renilla luciferase reporterplasmids were co-transfected into HEK293T cells with expression plasmidfor RIP1 and USP21 as well as increased amounts of expression plasmidfor Ubv.21.4 (SEQ ID NO: 7) or Ub.wt (SEQ ID NO: 1); 36 hours later,NF-κB activation in the cells was determined by measuring luciferaseactivity in the cell lysates. (E) Expression plasmids for RIP1 and USP21were co-transfected with increased amounts of expression plasmid forUbv.21.4 (SEQ ID NO: 7) or Ub.wt (SEQ ID NO: 1); 36 hours later, RIP1polyubiquitination was determined by immunoblotting of cell lysates withanti-RIP1 antibody. (F) Expression plasmid for vector control orMyc-USP21 was co-transfected into HEK293T cells with expression plasmidfor Flag-Ubv.21.4 (SEQ ID NO: 7) or Ub.wt (SEQ ID NO: 1); 36 hourslater, immunoprecipitations were performed with anti-Myc antibody forMyc-USP21, followed by immunoblotting with anti-Flag antibody forFlag-Ub.

DETAILED DESCRIPTION OF THE DISCLOSURE Definitions

The term “active site” as used herein refers to the surface exposedresidues of the substrate which are located in the substrate bindingsite of the enzyme or the substrate binding partner.

The term “analog” as used herein includes any active agent capable ofperforming the function of the enzyme inhibitors or substrate bindingpartners disclosed herein, and may include peptide mimetics and thelike.

The term “allosteric inhibition” as used herein refers to a change inthe shape of the active site of an enzyme or substrate binding partnerwhen an inhibitor binds to it. For example, an allosteric inhibitorchanges the shape of the active site such that the substrate is nolonger able to bind the enzyme or substrate binding partner.

The term “cofactor” as used herein refers to non-protein molecules thatmust be associated with certain enzymes for such enzymes to function.Cofactors can be inorganic compounds, such as metal ions or organiccompounds, such as flavin or heme.

The term “combinatorial library” as used herein refers to a collectionof substrate variants that have been randomized or mutated in the activesite.

The term “competitive inhibition” as used herein refers to competitionbetween the variant and the substrate for the enzyme or the substratebinding partner, for example, competition for binding of the enzymewhere only one can bind at a time.

A “conservative amino acid substitution” as used herein, is one in whichone amino acid residue is replaced with another amino acid residuewithout abolishing the desired function or activity of the enzymeinhibitors or substrate variants disclosed herein. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid, asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. Conserved amino acidsubstitutions involve replacing one or more amino acids of thepolypeptides of the disclosure with amino acids of similar charge, size,and/or hydrophobicity characteristics. When only conserved substitutionsare made the resulting molecule should be functionally equivalent.Changes which result in production of a chemically equivalent orchemically similar amino acid sequence are included within the scope ofthe disclosure. If the enzyme inhibitors or substrate variants of thepresent disclosure are made using recombinant DNA technology,conservative substituted variants of the enzyme inhibitors or substratevariants may be made by using polypeptide engineering techniques such assite directed mutagenesis, which are well known in the art forsubstitution of amino acids. For example, a hydrophobic residue, such asglycine can be substituted for another hydrophobic residue such asalanine. An alanine residue may be substituted with a more hydrophobicresidue such as leucine, valine or isoleucine. A negatively chargedamino acid such as aspartic acid may be substituted for glutamic acid. Apositively charged amino acid such as lysine may be substituted foranother positively charged amino acid such as arginine. The phrase“conservative substitution” also includes the use of a chemicallyderivatized residue in place of a non-derivatized residue provided thatsuch polypeptide displays the requisite activity.

The term “control” as used herein refers to a positive control such as aprotein known to inhibit the enzyme or a negative control such as aprotein known not to inhibit the enzyme, the wild type substrate or anabsence of inhibitor. The term also includes a predetermined standard.

The term “derivative” refers to a peptide having one or more residueschemically derivatized by reaction of a functional side group. Suchderivatized molecules include for example, those molecules in which freeamino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included asderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexample: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine. A derivative of a polypeptidealso optionally includes polypeptides comprising forms of amino acidsthat are oxidized.

The term “enzyme” as used herein refers to a protein or protein-basedmolecule that catalyzes a specific reaction in a living organism,converting a substrate or substrates into a product or products. Forexample, an enzyme provides a binding surface that mediates highspecificity for its substrate and associated cofactors and also allowsrelease of the reaction products. Enzymes regulate a wide variety ofprocesses in a living organism, including without limitation, signaltransduction, cell regulation, cell movement, cell death and proteindegradation. Typical enzymes include transferases, anhydrases,synthases, synthetases, polymerases, hydrolases, such as proteases,lyases, and ligases. In various aspects of the disclosure, the term“enzyme” includes ubiquitin-specific proteases (USPs) and ubiquitinligases.

The term “enzyme-substrate complex” as used herein refers to the complexformed when a substrate molecule binds with the active site of an enzymeand optionally, a cofactor. In addition, the term “substrate bindingpartner-substrate complex” as used herein is formed when a substratemolecule binds with the active site of a protein interacting motif,which in some aspects may be parts of larger proteins. For example, inmore particular aspects, ubiquitin or ubiquitin variants interact withubiquitin interaction motifs (UIM).

The term “fragment” as used herein means a portion of a polypeptide thatcontains at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or more of the entire length of thereference polypeptide.

The term “isolated and purified” as used herein refers to a nucleic acidor amino acid substantially free of cellular material or culture mediumwhen produced by recombinant DNA techniques, or chemical precursors, orother chemicals when chemically synthesized. An “isolated and purified”nucleic acid is also substantially free of sequences which naturallyflank the nucleic acid (i.e. sequences located at the 5′ and 3′ ends ofthe nucleic acid) from which the nucleic acid is derived.

The term “nucleic acid” as used herein refers to a sequence ofnucleotide or nucleoside monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted sequences comprising non-naturally occurringmonomers or portions thereof, which function similarly. The nucleic acidsequences of the present disclosure may be ribonucleic (RNA) ordeoxyribonucleic acids (DNA) and may contain naturally occurring basesincluding adenine, guanine, cytosine, thymidine and uracil. Thesequences may also contain modified bases such as xanthine,hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl, and other alkyladenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosineand 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thio-alkyl adenines, 8-hydroxyl adenine andother 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiolguanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other8-substituted guanines, other aza and deaza uracils, thymidines,cytosines, adenines, or guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine.

The term “product” as used herein refers to the resulting compound orcompounds produced by an enzyme from its substrate.

The term “substrate” as used herein refers to a molecule at thebeginning of an enzymatic reaction i.e. the substance that is acted uponby an enzyme or binds to a substrate binding partner. In some aspects,the term substrate is used herein to identify ubiquitin or a ubiquitinvariant.

The terms “transformed with”, “transfected with”, “transformation” and“transfection” are intended to encompass introduction of nucleic acid(e.g. a vector) into a cell by one of many possible techniques known inthe art. The term “transformed host cell” as used herein is intended toalso include cells capable of glycosylation that have been transformedwith a recombinant expression vector of the disclosure. Prokaryoticcells can be transformed with nucleic acid by, for example,electroporation or calcium-chloride mediated transformation. Forexample, nucleic acid can be introduced into mammalian cells viaconventional techniques such as calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran mediated transfection, lipofectin,electroporation or microinjection. Suitable methods for transforming andtransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor LaboratoryPress, 2001), and other laboratory textbooks.

The term “ubiquitin” or “Ub” as used herein refers to ubiquitin from anyspecies or source and includes the full-length protein as well asfragments or portions of the protein. Human ubiquitin has the amino acidsequence as shown in SEQ ID NO: 1 and has the Genbank Accession No. forpoly-ubiquitin of: BAA23486 and for the ubiquitin isoform C of:EAX04505.

The term “ubiquitin variant” or “Ubv” as used herein refers to aubiquitin polypeptide that comprises at least one amino acidsubstitution, deletion, insertion, addition, or modification withrespect to the native or wildtype (wt) ubiquitin polypeptide of SEQ IDNO: 1. In some aspects, a ubiquitin variant, Ub.wt variant, comprisingtwo additional C-terminal amino acids (two glycines at positions 76a and76b, i.e., amino acids 77 and 78 of SEQ ID NO: 21) is used in themethods described herein to identify ubiquitin enzyme inhibitors orubiquitin variants that bind ubiquitin interaction motifs.

The term “ubiquitin enzyme” as used herein refers to an enzyme whosesubstrate is ubiquitin and includes, without limitation, Ub-activatingenzymes (E1 enzymes), Ub-conjugating enzymes (E2 enzymes), Ub ligase (E3enzymes) and deubiquitinating enzymes (DUBs).

Compounds of the Disclosure

Exemplary ubiquitin inhibitors are provided in Tables 1-10.

TABLE 1 UBIQUITIN INHIBITORS WITH BINDING SPECIFICITY TO USP8 and USP21SEQ UBIQUITIN ID VARIANT NO SEQUENCE Ubv.8.2 2MRIVVKTLMGRTIILEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIHNHSALYLLLKLRGG Ubv.8.3 3 MYIFVKTFMGRTIYLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN ISKVSALYLLFRLRGG Ubv.21.1 4MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKISTLFLLMRLRGG Ubv.21.2 5 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN ILKRSTLFLLLKLRGG Ubv.21.3 6MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNILKSSTLFLLLRLRGG Ubv.21.4 7 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKWSTLFLLLRLRGG Ubv.21.5 8MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKDSTLFLLLRLRGG

TABLE 2 UBIQUITIN INHIBITORS WITH BINDING SPECIFICITY TO USP2 SEQUBIQUITIN ID VARIANT NO SEQUENCE USP2-A1 22MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVMRLWSHRR USP2-A10 23 MQIFVNTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLWSEGT USP2-A12 24MQIFVMTLTGQNITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLLSGGH USP2-A3 25 MQIFVKTLWTRTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLYGVKR USP2-A6 26MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLYGQAA USP2-A7 27 MDIFVNTITGKIITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IPRYSTLHLLLRLRGEIQ USP2-A9 28MQIFVMTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKYSTLHLVLRLLGVSH USP2-B10 29 MQIFVYTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLYSLDR USP2-B11 30MHIFVNTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLWSCKG USP2-B12 31 MQIFVKTLSGRTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLLSGRK USP2-B7 32MQIFVNTLTGTHITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGGG USP2-B8 33 MQIFVMTLTGKHITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLRGGGG USP2-C10 34MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLHSNAM USP2-C11 35 MQIFVKTIAGKAITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLLSGKR USP2-C3 36MQIFVNTLSGKHITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGGG USP2-C5 37 MQIFVNTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLFSARV USP2-C7 38MQIFVNTLTGRHITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGGG USP2-C8 39 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IHKESTLHLVLRLLSGRR USP2-C9 40MQIFVNTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIKKESTLHLVLRLWSWRR USP2-D1 41 MQIFVNTLTARNITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLFLVLRLRSENL USP2-D11 42MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLLSGSI USP2-D3 43 MDIFVNTLTGNTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLRGGGG USP2-D4 44MEIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLWSISF USP2-D6 45 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLLSRSK USP2-D7 46MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLLGYSK USP2-D9 47 MNIFVKTLAGNHITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLRGGGG

TABLE 3 UBIQUITIN INHIBITORS WITH BINDING SPECIFICITY TO USP5 SEQUBIQUITIN ID VARIANT NO SEQUENCE USP5-E4 48MEIYVNTRLLETINLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKKSTLYLVLRLLGQRH USP5-F11 49 MQIFVQTRVMNTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFATKQLEDGRTLSDYN IHKDSTLHLLLRMRRLNK USP5-F3 50MQIFVQTRAMWTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIPKESTLHLVLRLRGGGG USP5-F7 51 MQIFVNTRTMFTIRLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLRGGGG USP5-F8 52MRIFVKTRMMESIFLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGQQLEDGRTLSDYNIQKESTLHLVVRFRSARR USP5-F9 53 MPIFVNTRWMKTIPLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFDRKQLEDGRTLSDYN IQKESTLHLVLRDLGVFC USP5-G10 54MQIMVHTRVMNSITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGGG USP5-G11 55 MQISVNTRMMESITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN ILPESTLHLVLRLRGGGG USP5-G3 56MQIVVNTRMMETITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGGG USP5-G4 57 MHIFVNTRIMETITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLRGGGG USP5-G5 58MHISVNTRSMWTIQLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGGG USP5-G6 59 MQIFVHTRMMETITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IHKESTLHLVLRLRGGGG USP5-G8 60MQIIVRTRAMWTIALEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIRTESTLHLVLRLRGGGG USP5-G9 61 MQIFVNTRMMDSIMLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLRGGGG USP5-H10 62MQIFVNTRMMMTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGGG USP5-H11 63 MQIVVNTRSMNTIHLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKRSTLYLVLRLRGGGG USP5-H12 64MRIVVNTRYMNTISLEVEPSDTIENVKAKI QDKEGIPPDQQTLIFAGKQLEDGRTLSDYNIHKESTLHLVLRLRGGGG USP5-H4 65 MQIFVNTRLMYSITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRISRGLP USP5-H7 66MQILVRTRIMETITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGNQLEDGRTLSDYNIQKESTLHLVLRLRGGGG USP5-H8 67 MQIFVNTRMMETIALEVEPSDTIENVKAKIQDKEGIPPDQQSLIFAGKRLEDGRTLSDYN IQKSSTLHLVLRLRGGGG USP5-H9 68MQIFVNTRMMNTIDLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGGG

TABLE 4 UBIQUITIN INHIBITORS WITH BINDING SPECIFICITY TO USP10 SEQUBIQUITIN ID VARIANT NO SEQUENCE Ubv.10.1 69MQIFVETPMGKTIALEVEPSDTIENVKAKI QDKEGIPPDQQRLVFVGKLLEDGRTLSDYNIQRWSTLALKFRLLAKNL

TABLE 5 UBIQUITIN INHIBITORS WITH BINDING SPECIFICITY TO USP48 SEQUBIQUITIN ID VARIANT NO SEQUENCE USP48-Ubv.48.01 70MQIIVKTLTGGTIGLEVEPSDTIENVKAKI QDKEGIPPDQQRLFFAFTPLEDGRTLSDYNIQMESTLRLFLRPRGLKE USP48-Ubv.48.02 71 MQISVKTLIGNMITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFVFKQLEDGRTLSDYN IGKHSTLHLWLRLLGIGK USP48-Ubv.48.03 72MQIFVKTLNGNIISLEVEPSDTIENVKAKI QDKEGIPPDQQRLFFAWKQLEDGRTLSDYNIPKESTLPLFLRLRGGGG USP48-Ubv.48.04 73 MQISVKTLTGGTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLFFAFTPLEDGRTLSDYN IGKQSTLHLWLRLLGLEE USP48-Ubv.48.05 74MRIFVETLTGQIINLEVEPSDTIENVKAKI QDKEGIPPDQQRLNFAGKLLEDGRTLSDYNIKKWSTLHLVLRLRGGGG

TABLE 6 UBIQUITIN INHIBITORS WITH BINDING SPECIFICITY TO NEDD4 SEQUBIQUITIN ID VARIANT NO SEQUENCE Nedd4-N4-A11 75MLILVKTLTGHSITLEVEPSDTIENVKAKI QDKEGIPPDQQRLFFAGKQLEDGRTLSDYNIHKFSTLHLVKRLRGRWK Nedd4-N4-A9 76 MRIFVTTLTGRAITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFTGKKLEDGRTLSDYN IPKESTLYLVKRLRGMEQ Nedd4-N4-B1 77MHIFVRTLTRKIITLEVEPSDTIENVKAKI QDKEGIPPDQQRLFFTGMTLEDGRTLSDYNIQKESTLHLVLRLRGGGG Nedd4-N4-B10 78 MRILVKTLTRKVITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGQRLEDGRTLSDYN IQKESTLHLVLRLRGGGG Nedd4-N4-B4 79MQIFVKTMRRESISLEVEPSDTIENVKAKI QDKEGIPPDQQRLFFTGKQLEDGRTLSDYNIQKESTLHLVKRLPGRQY Nedd4-N4-B5 80 MQIVVKTLSRRTIGLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKKLEDGRTLSDYN IQKESTLHLALKLPGMGG Nedd4-N4-B6 81MQIFVKTFTGKSITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFDRNKLEDGRTLSDYNIKKESTLGLVLRLRGGGG Nedd4-N4-C1 82 MQILVQTLTRKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLFFAGTQLEDGRTLSDYN IQKHSTLYLVLRLLGRRH Nedd4-N4-C10 83MQILVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGLKLEDGRTLSDYNIQKVSTLYLVKTFPGRRQ Nedd4-N4-C11 84 MQIFVKTLAGWGITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IRYDSTLHLVGRLRGGGG Nedd4-N4-C12 85MQILVKTLARTSIILEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGGG Nedd4-N4-C2 86 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKDSTLHLVRRLPRMGK Nedd4-N4-C4 87MYISVKTLTGESISLEVEPSDTIENVKAKI QDKEGIPPDQQKLFFAGKILEDGRTLSDYNILKWSTLHLVKRLRAVHM Nedd4-N4-C5 88 MQIFVKTLTRKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKRLEDGRTLSDYN IQKESTLHLVLRLRGGGG Nedd4-N4-C6 89MPIVVKTLAGYTIHLEVEPSDTIENVKAKI QDKEGIPPDQQKLLFAGNRLEDGRTLSDYNIHNGSTLYLVKRLRGGGG Nedd4-N4-D1 90 MQIFVNTLARTSISLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLPGTKV Nedd4-N4-D10 91MQIFVRTLMRKSISLEVEPSDTIENVKAKI QDKEGIPPDQQKLIFAGKLLEDGRTLSDYNIQKESTLHLVKKLILLRE Nedd4-N4-D11 92 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLRGGGG Nedd4-N4-D12 93MQIYVKTLTRKRITLEVEPSDTIENVKAKI QDKEGIPPDQQKLFFNGKQLEDGRTLSDYNIQKESTLHLVLRLRGGGG Nedd4-N4-D2 94 MQIYVKTLTRKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFVGKQLEDGRTLSDYN IQKESTLYLVLRLRGGGG Nedd4-N4-D3 95MKISVNTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKYSTLYLVKRLRRLKQ Nedd4-N4-D5 96 MQIFVPTLVQKAINLEVEPSDTIENVKAKIQDKEGIPPDQQRLFFFRKPLEDGRTLSDYN IKKESTLHLLWRFSSRLM Nedd4-N4-D8 97MQIFVRTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAKQQLEDGRTLSDYNILKESTLGLVRRLRGLVS Nedd4-N4-D9 98 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQQVSTLYLVKRLRGGGG

TABLE 7 UBIQUITIN INHIBITORS WITH BINDING SPECIFICITY TO ITCH SEQUBIQUITIN ID VARIANT NO SEQUENCE Ubv.ITCH.01 99MPILVKTLRGQSIILEVEPSDTIENVKAKI QDKEGIPPDQQFLIFARKHLEDGRTLSDYNIQKRSTLYLFLRFHGMVA Ubv.ITCH.02 100 MHILVKTLRGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLLFGGNKLEDGRTLSDYN IQKESTLYLLLRRLGSKF Ubv.ITCH.03 101MQIFVITHTWRTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLLFARQKLEDGRTLSDYNIQKDSTLHLVLIRRVSKR Ubv.ITCH.04 102 MQIFVKTLTGLSITLEVEPSDTIENVKAKIQDKEGIPPDQQILIFGGKRLEDGRTLSDYN IQKKSTLYLLMRLRGVSR Ubv.ITCH.05 103MQIFVKTLTGTDITLEVEPSDTIENVKAKI QDKEGIPPDQQILLFAGKQLEDGRTLSDYNIPGDSTLYLLMRFGVNKR Ubv.ITCH.06 104 MPILVQTLRGQSIILEVEPSDTIENVKAKIQDKEGIPPDQQFLIFARTHLEDGRTLSDYN IQKGSTLYLLLRFHGTVA

TABLE 8 UBIQUITIN INHIBITORS WITH BINDING SPECIFICITY TO UIM SEQUBIQUITIN ID VARIANT NO SEQUENCE Ubv.UIM1.01 105MQILVKTVIVKTISLEVEPSDTIENVKAKI QDKEGIPPDQQTLIFAGMQLEDGRTLSDYNIRRASTLHLVGRLRGGGG Ubv.UIM1.02 106 MQIFVQTLIVKTIILEVEPSDTIENVKAKIQDKEGIPPDQQYLIFAGKQLEDGRTLSDYN IGMESTLRLVGRLRGGGG Ubv.UIM1.03 107MPISVVTLIVKTITLEVEPSDTIENVKAKI QDKEGIPPDQQSLIFAGKQLEDGRTLSDYNIQNRSTLHLVGRLRGGGG Ubv.UIM1.04 108 MLIFVTTVRVSTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN ILKESTLKLVWIFRGNRT Ubv.UIM1a.01 109MQILVKTVIVKTISLEVEPSDTIENVKAKI QDKEGIPPDQQTLIFAGMQLEDGRTLSDYNIRRASTLHLVGRLRGGGG Ubv.UIM1a.02 110 MEIVVQTLIVKSITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFGGDFLEDGRTLSDYN IQKESTLHLVSWLRGGGG Ubv.UIM1a.03 111MQIIVETITVKTIALEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIHKESTLLLVSWLHGDRQ Ubv.UIM1a.04 112 MQIFVQTLIVKTIILEVEPSDTIENVKAKIQDKEGIPPDQQYLIFAGKQLEDGRTLSDYN IGMESTLRLVGRLRGGGG Ubv.UIM1a.05 113MQIFVQTITVMRIALEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGMQLEDGRTLSDYNIKRDSTLYLVSSLRGLRA Ubv.UIM1a.06 114 MQIFVQTLTVKSITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGRQLEDGRTLSDYN IQKGSTLHLVAWLRGGGG Ubv.UIM1b.01 115MRIFVKTITVKSIHLEVEPSDTIENVKAKI QDKEGIPPDQQTLIFAGKLLEDGRTLSDYNIKKESTLHLVAKHRGMEV Ubv.UIM1b.02 116 MQIFVKTLTVTTIYLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKDSTLGLVLKFRALVR Ubv.UIM1b.03 117MQIFVITFSGRTITLEVEPSDTIENVKAKI QDKEGIPPDQQNLIFGGRQLEDGRTLSDYNIQQVSTLLLVLSLRGTRE Ubv.UIM1b.04 118 MQIFVKTLAVKTIELEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKDSTLGLVLSVRVLRR Ubv.UIM1b.05 119MQINVNTLMVKAITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFGAKQLEDGRTLSDYNIQKESTLYLVFSLRGKGQ Ubv.UIM1b.06 120 MLIFVKTLRVETITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IGKESTLGLVTRLRVYES Ubv.UIM1b.07 121MTIFVTTIIVNTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIKKESTLRLVSRHRRTVK Ubv.UIM1b.08 122 MHIFVKTLTVKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFVRQHLEDGRTLSDYN IQKESTLGLVLSLRGIAK Ubv.UIM1b.09 123MQIFVGTLTVNGINLEVEPSDTIENVKAKI QDKEGIPPDQQRLFFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGGG Ubv.UIM1b.10 124 MQIFVKTLTVNTITLEVEPSDTIENVKAKIQDKEGIPPDQQKLIFAGKQLEDGRTLSDYN IQKDSTLGLVSRLRGGGG Ubv.UIM1b.11 125MQILVTTIIVRTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFSGKELEDGRTLSDYNIRNKSTLYLVGRLRGGGG Ubv.UIM1b.12 126 MQIFVNTLRAKFITLEVEPSDTIENVKAKIQDKEGIPPDQQRLLFAGQQLEDGRTLSDYN IHKESTLSLVASLRGDQK Ubv.UIM1b.13 127MQIFVLTLKWKTIALEVEPSDTIENVKAKI QDKEGIPPDQQRLIFSGWHLEDGRTLSDYNIEKGSTLGLVLTLRGGGG Ubv.UIM1b.14 128 MEISVKTLAVKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLFFAGKLLEDGRTLSDYN IHRESTLLLVLRIYRRAS Ubv.UIM1b.15 129MKIFVTTLTVKTIALEVEPSDTIENVKAKI QDKEGIPPDQQGLIFAGKQLEDGRTLSDYNIQKESTLLLGVMLRANRS

TABLE 9 UBIQUITIN INHIBITORS WITH BINDING SPECIFICITY TO USP8 SEQUBIQUITIN ID VARIANT NO SEQUENCE USP8-A01 130MQIFVKTPKGKNITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFIAKHLEDGRTLSDYNIPKESTLSLLMSFPRTVR USP8-A03 131 MQIFVKTLRGHLITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFVAKQLEDGRTLSDYN IHKESTLQLMFKPRGQRR USP8-A04 132MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQSLSFAGKPLEDGRTLSDYNIHRTSTLHLVFSFRSRVK USP8-A06 133 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQSLSFAGKPLEDGRTLSDYN IHRTSTLHLVFSFRSKVK USP8-A07 134MQIFVKTLRGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLFFDVKQLEDGRTLSDYNIQKESTLDLFLGLRGRSR USP8-A08 135 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFSGKPLEDGRTLSDYN IPKESTLDLGLRRRTTVS USP8-A09 136MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNINWESTLRLWSRVRGKYQ USP8-B03 137 MQIFVKTVKGETIVLEVEPSDTIENVKAKIQDKEGIPPDQQKLYFDVKQLEDGRTLSDYN IQQESTLHLLVRLGGRGG USP8-B06 138MQIFVITFPGKTIALEVEPSDTIENVKAKI QDKEGIPPDQQRLIFDVKQLEDGRTLSDYNIQNESTLNLVWRPRGVLN USP8-B09 139 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFDVKQLEDGRTLSDYN IQTYSTLHLGLRLRGHTT USP8-B10 140MQIFVKTLMGRAITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIHKASTLYLMLRLRGGGG

TABLE 10 UBIQUITIN INHIBITORS WITH BINDING SPECIFICITY TO USP21 SEQUBIQUITIN ID VARIANT NO SEQUENCE USP21-E10 141MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIEWKSTLFLLLRLRSILL USP21-E11 142 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IPKESTLFLLVRLFVKQI USP21-E12 143MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIETGSTLFLLFRLRGMGT USP21-E2 144 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKLSTLFLLLRFRSYMR USP21-E3 145MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIYHKSTLFLLVKFRGLTP USP21-E4 146 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQNESTLFLLLRFPRVQA USP21-E6 147MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIPKASTLFLLLRLHAQRR USP21-E7 148 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKFSTLFLLLRLGGWYL USP21-E9 149MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKQSTLFLVLRLRGKDM USP21-F11 150 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQRQSTLYLLIRIHRRKR USP21-F12 151MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKHSTLYLLFRFTVKGR USP21-F2 152 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IPRHSTLFLLFRLRDTSR USP21-F3 153MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIHKASTLFLVLRVRAHKQ USP21-F5 154 MEIFVKTLSGMTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKHSTLFLVLRLHVGNN USP21-F6 155MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKKSTLFLVLRLHSTRE USP21-F9 156 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IHKESTLFLLLRFRGTLS USP21-G11 157MQIFVKTVTGRSITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQTKSTLFLVLRFRGNTR USP21-G2 158 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IEKESTLFLLLRLPSWKG USP21-G3 159MQIYVKTLPGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIHLRSTLFLLFRPRRLYK USP21-G4 160 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IEKKSTLFLLLRLYWEDK USP21-G5 161MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIYNESTLFLLARLRFGRA USP21-G6 162 MQIFVKTSTGRTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQRQSTLFLIWRLTSAMV USP21-G9 163MQIFVKTHTAKTILLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNINLESTLFLLFRFRGNTL USP21-H1 164 MQIFVKTPTGMSITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKVSTLFLVFKLWRRSM USP21-H3 165MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIHKESTLFLLLRLSWDFK USP21-H5 166 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IHKHSTLFLLLRLRSRLK USP21-H6 167MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIVKNSTLFLLLRIHGSQK USP21-H7 168 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IHKQSTLFLVLRLRGLSS USP21-H8 169MQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKDSTLFLLLRLRGLQYMethods

The present disclosure demonstrates that by enhancing the bindingproperties of ubiquitin (Ub) to existing natural ubiquitin binding sitesof ubiquitin-specific-proteases (USP) the function of these enzymes wasinhibited. To do so a combinatorial library of Ub variants was createdby genetically engineering diversity in the interface region buried bythe interaction of both proteins. As a proof of concept using this novelmethod, site specific inhibitors to USP8 and USP21 were created.

Accordingly, the present disclosure provides a method of producingenzyme-specific inhibitors comprising:

(a) identifying active site residues of a substrate in anenzyme-substrate complex;

(b) randomizing the active site residues to produce a combinatoriallibrary of substrate variants; and

(c) selecting substrate variants that inhibit enzyme activity.

Identification of active site residues is performed by using variousmethods known in the art, including without limitation, analysis of 3Dstructures, alanine scanning, shotgun scanning and other mutationalanalysis of interaction interfaces.

In one embodiment, randomizing the active site residues in b) is bygenetic engineering. For example, variants can be created where eachactive site residue is replaced with a different amino acid, such that alibrary of variants comprising all 20 amino acids in each active siteposition is produced. In one embodiment, randomization is performed toyield 75% wild type amino acid residues and 25% mutated amino acidresidues at the active site residues. For ubiquitin, the active siteresidues comprise regions 1-3 as shown in FIG. 1.

The combinatorial library can be screened for enzymatic inhibition, forexample, by phage display, mRNA display, ribosome display, yeast displayor other similar technologies to determine the inhibition of enzymeactivity compared to a control. In one embodiment, the control is adifferent enzyme to test for enzyme specificity. In another embodiment,the control is the wild type enzyme to test for amount of inhibition.

Enzyme activity can be inhibited in a variety of ways. In oneembodiment, the substrate variant has a higher affinity to the activesite of the enzyme, which provides competitive inhibition. In anotherembodiment, the substrate variant blocks the active site of the enzymeafter an enzymatic reaction, which provides product inhibition. In yetanother embodiment, the substrate variant induces a change in the shapeof the enzymatic active site, which provides allosteric inhibition.

The enzyme can be any enzyme, for example, a transferase, hydrolase,lyase, or ligase.

In one embodiment, the substrate is ubiquitin and the enzyme is aUb-activating enzyme (E1 enzyme), a Ub-conjugating enzymes (E2 enzyme)or a Ub ligase (E3 enzyme). In one embodiment, the Ub ligase is a HECTubiquitin Ligase, such as NEDD4 or ITCH. In another embodiment, thesubstrate is ubiquitin and the enzyme is a deubiquitinating enzyme. Inone embodiment, the deubiquitinating enzyme is a ubiquitin specificprotease (USP), a ubiquitin C-terminal hydrolase (UCH), an ovarian tumorprotease (OTU), a Machado-Joseph disease protease (MJD) or aJAB1/MPN/MOV34 metalloenzyme (JAMM). In one embodiment, thedeubiquitinating enzyme is a ubiquitin specific protease (USP). In anembodiment, the USP is USP2, USP4, USP5, USP7, USP8, USP9x, USP9y,USP10, USP12, USP14, USP16, USP21, USP28, USP42, USP46 or USP48. In oneembodiment, the USP is USP2, USP5, USP8, USP10, USP21, or USP48. In afurther embodiment, the USP includes all splice forms, i.e. isoforms, ofUSP2, USP4, USP5, USP7, USP8, USP9x, USP9y, USP10, USP12, USP14, USP16,USP21, USP28, USP42, USP46 or USP48.

In another embodiment, the substrate is ubiquitin as shown in SEQ ID NO:1 and the resulting variant comprises mutations in the active siteresidues present in region 1 (amino acids 2-14) and region 3 (aminoacids 62-72). In yet another embodiment, the substrate is ubiquitin asshown in SEQ ID NO: 1 and the resulting variant comprises mutations inthe active site residues present in region 3 (amino acids 62-72). In yetanother embodiment, the substrate is ubiquitin and the resulting variantcomprises mutations in at least one of positions 2, 4, 6, 8, 9, 10, 11,12, 14, 35, 37, 39, 40, 42, 44, 46, 47, 48, 49, 62, 63, 64, 66, 68, 70,71 or 72, optionally in at least one of positions 2, 4, 6, 8, 9, 10, 11,12, 14, 62, 63, 64, 66, 68, 70, 71 or 72 or in at least one of positions62, 63, 64, 66, 68, 70, 71 or 72. In another embodiment, the resultingvariant comprises mutations in at least 2, 4, 6 or 8 of the positions.

In another embodiment, the substrate is a ubiquitin variant as shown inSEQ ID NO: 21 and the resulting inhibitors comprises an amino acidmutation in region 1 (amino acids 2-14), region 2 (amino acids 42-49),or region 3 (amino acids 62-78) in the amino acid sequence of aubiquitin variant (Ubv) as set forth in SEQ ID NO: 21. In yet anotherembodiment, the substrate is ubiquitin and the resulting inhibitorscomprises an amino acid mutation at position 2, 4, 6, 8, 9, 10, 11, 12,14, 42, 44, 46, 47, 48, 49, 62, 63, 64, 68, 70, 71, 72, 73, 74, 75, 76,77, or 78 of the amino acid sequence of a ubiquitin variant (Ubv) setforth in SEQ ID NO: 21.

Enzyme Inhibitors

The methods described herein are applied, by way of example, on theprotein substrate ubiquitin and enzymes in the ubiquitination anddeubiquitination pathway.

Within the ubiquitination-pathway multiple enzymes rely on the efficientrecognition of ubiquitin and ubiquitin chains as their primarysubstrate. By modifying ubiquitin as protein substrate in theabove-described manner, enzymes in the ubiquitin-pathway can beinhibited. Target molecules in the ubiquitination pathway for anUb-based inhibitor are Ub-activating enzymes (E1 enzymes),Ub-conjugating enzymes (E2 enzymes) and Ub ligases (E3 enzymes). Theseenzymes are responsible for attaching ubiquitin to other proteins and bythat influence the homeostasis of proteins, their localization withinthe cell and regulate cellular signalling. The human genome encodes for2 Ub-activating E1 enzymes while at least 38 conjugating E2-enzymes areknown to date [Ye and Rape 2009]. Additionally, more than 600

E3 enzymes have been identified so far and can be divided into 2 majorfamilies: HECT domain—E3 ligases and RING-domain E3-ligases. More than90% of the E3 ligases belong to the RING domain ligase family which mainfunctions are to interact with Ub conjugated E2's and the correspondingsubstrate. RING domain E3 ligases facilitate the transfer of Ub directlyfrom the E2 enzyme to the substrate. The chain topology found inpoly-ubiquitinated proteins often depend on the involved E2 enzyme.Therefore, Ub based inhibitors of E2-conjugating enzymes can be used tointerfere and control multiple targets at once due to their involvementin many different aspects of ubiquitination. In contrast to RING domainE3 ligases, HECT domain E3 ligases covalently interact through athio-ester bond with Ub and transfer Ub to the substrate without theinvolvement of E2-conjugating enzymes. Therefore, targeting HECT-domainE3-ligases with Ub-based inhibitors would allow a more direct andlimited interference with ubiquitylated substrates. In summary, multiplelevels of intracellular control are conceivable by targeting theubiquitination machinery either through inhibiting E2 or E3 enzymes.Another group of target molecules are deubiquitinating enzymes such asUbiquitin specific proteases (USPs), Ubiquitin C-terminal hydrolases(UCHs), ovarian tumor proteases (OTUs), Machado-Joseph disease proteases(MJDs) and JAB1/MPN/MOV34 metalloenzymes (JAMMs) [Ventii and Wilkinson2008; Komander, Clague et al. 2009; Reyes-Turcu, Ventii et al. 2009].Deubiquitinating enzymes provide a large binding surface for therecognition and subsequent proteolysis of mono-ubiqutinated andpoly-ubiquitinated proteins. In poly-ubiquitinated proteins Ub canappear in different conformations such as K6, K11, K27, K29, K33, K48,K63 linked Ub-chains as well as linear Ub-chains.

Accordingly, the present disclosure provides a ubiquitin enzymeinhibitor comprising mutations in region 3 (amino acids 62-72) of theamino acid sequence of ubiquitin as shown in SEQ ID NO: 1 andoptionally, further comprising mutations in region 1 (amino acids 2-14)of the amino acid sequence of ubiquitin as shown in SEQ ID NO: 1.

In another embodiment, the ubiquitin enzyme inhibitor comprisesmutations in at least one of positions 2, 4, 6, 8, 9, 10, 11, 12, 14,35, 37, 39, 40, 42, 44, 46, 47, 48, 49, 62, 63, 64, 66, 68, 70, 71 or 72of the amino acid sequence of ubiquitin as shown in SEQ ID NO:1,optionally in at least one of positions 2, 4, 6, 8, 9, 10, 11, 12, 14,62, 63, 64, 66, 68, 70, 71 or 72 of the amino acid sequence of ubiquitinas shown in SEQ ID NO:1 or in at least one of positions 62, 63, 64, 66,68, 70, 71 or 72 of the amino acid sequence of ubiquitin as shown in SEQID NO:1. In another embodiment, the ubiquitin enzyme inhibitor comprisesmutations in at least 2, 4, 6 or 8 of the positions.

The present disclosure also provides a ubiquitin enzyme inhibitorcomprising an amino acid mutation in region 1 (amino acids 2-14), region2 (amino acids 42-49), or region 3 (amino acids 62-78) of the amino acidsequence of a ubiquitin variant (Ubv) set forth in SEQ ID NO: 21.

In one embodiment, a ubiquitin enzyme inhibitor of the disclosurecomprises an amino acid mutation at position 2, 4, 6, 8, 9, 10, 11, 12,14, 42, 44, 46, 47, 48, 49, 62, 63, 64, 68, 70, 71, 72, 73, 74, 75, 76,77, or 78 in the amino acid sequence of the ubiquitin wildtype variant(Ubv) polypeptide set forth in SEQ ID NO: 21.

In one embodiment, the enzyme is a Ub-activating enzyme (E1 enzyme), aUb-conjugating enzymes (E2 enzyme) or a Ub ligase (E3 enzyme). In oneaspect, the E3 ligase is Nedd4 or ITCH. In another embodiment, theenzyme is a deubiquitinating enzyme. In one embodiment, thedeubiquitinating enzyme is a ubiquitin specific protease (USP), aubiquitin C-terminal hydrolase (UCH), an ovarian tumor protease (OTU), aMachado-Joseph disease protease (MJD) or a JAB1/MPN/MOV34 metalloenzyme(JAMM).

In an embodiment, the deubiquitinating enzyme is a USP. In oneembodiment, the USP inhibitor comprises a mutation at position 68optionally Tyr or Phe for His of the amino acid sequence of ubiquitin asshown in SEQ ID NO: 1. In another embodiment, the USP inhibitorcomprises a Val to Leu mutation at position 70 of the amino acidsequence of ubiquitin as shown in SEQ ID NO: 1.

In another embodiment, the USP is USP8 or USP21. In one embodiment, theUSP8 inhibitor comprises the sequence shown in SEQ ID NO: 2 (Ub variant8.2) or SEQ ID NO: 3 (Ub variant 8.3). In another embodiment, the USP21inhibitor comprises the sequence shown in SEQ ID NO:4 (Ub variant 21.1),SEQ ID NO:5 (Ub variant 21.2), SEQ ID NO:6 (Ub variant 21.3), SEQ IDNO:7 (Ub variant 21.4) or SEQ ID NO:8 (Ub variant 21.5).

In additional embodiments, the USP is USP2, USP5, USP8, USP10, USP21, orUSP48. In one embodiment, the USP inhibitor inhibits USP2 and comprisesany of the amino acid sequences of SEQ ID NOS: 22-47. In anotherembodiment, the USP inhibitor inhibits USP5 and comprises any of theamino acid sequences of SEQ ID NOS: 48-68. In another embodiment, theUSP inhibitor inhibits USP10 and comprises the amino acid sequence ofSEQ ID NO: 69. In another embodiment, the USP inhibitor inhibits USP48and comprises any of the amino acid sequences of SEQ ID NOS: 70-74. Inanother embodiment, the USP inhibitor inhibits USP8 and comprises any ofthe amino acid sequences of SEQ ID NOS: 130-140. In another embodiment,the USP inhibitor inhibits USP21 and comprises any of the amino acidsequences of SEQ ID NOS: 141-169.

In another embodiment, the E3 ubiquitin ligase is Nedd4 or ITCH. In oneembodiment, the Nedd4 binder comprises any of the amino acid sequencesof SEQ ID NOS: 75-98. In another embodiment, the ITCH binder comprisesany of the amino acid sequences of SEQ ID NOS: 99-104.

In another embodiment, the UIM binder comprises any of the amino acidsequences of SEQ ID NOS: 105-129.

The enzyme inhibitors disclosed herein also include conservativesubstitutions, as well as deletions and or additions that work insubstantially the same way as an inhibitor of ubiquitin. In oneembodiment, the enzyme inhibitor is a fragment of an amino acid sequencedisclosed herein that performs substantially the same function insubstantially the same way.

The enzyme inhibitors disclosed herein also include analogs andderivatives thereof.

The enzyme inhibitors may also contain or be used to obtain or design“peptide mimetics.” Peptide mimetics include synthetic structures thatmay serve as substitutes for peptides in interactions between molecules(see Morgan and Gainor. (1989), Ann. Reports Med. Chem. 24:243-252 for areview). Peptide mimetics include synthetic structures which may or maynot contain amino acids and/or peptide bonds but are designed to retainthe desired structural and functional features and thus may be suitablesubstitutes of the peptide inhibitor analog disclosed herein.

Peptide mimetics also include molecules incorporating peptides intolarger molecules with other functional elements (e.g., as described inWO 99/25044). Peptide mimetics also include peptoids, oligopeptoids(Simon et al (1972) Proc. Natl. Acad. Sci. USA 89:9367), and peptidelibraries containing peptides of a designed length representing allpossible sequences of amino acids corresponding to an isolated peptideof the disclosure. Peptide mimetics may be designed based on informationobtained by systematic replacement of L-amino acids by D-amino acids,replacement of side chains with groups having different electronicproperties, and by systematic replacement of peptide bonds with amidebond replacements. Local conformational constraints can also beintroduced to determine conformational requirements for activity of acandidate peptide mimetic. The mimetics may include isosteric amidebonds, or D-amino acids to stabilize or promote reverse turnconformations and to help stabilize the molecule. Cyclic amino acidanalogues may be used to constrain amino acid residues to particularconformational states. The mimetics can also include mimics of inhibitorpeptide secondary structures. These structures can model the3-dimensional orientation of amino acid residues into the knownsecondary conformations of proteins. Peptoids may also be used which areoligomers of N-substituted amino acids and can be used as motifs for thegeneration of chemically diverse libraries of novel molecules.

The disclosure further provides nucleic acids encoding the ubiquitinenzyme inhibitors disclosed herein.

Accordingly, the present disclosure provides a nucleic acid moleculeencoding a ubiquitin enzyme inhibitor comprising mutations in region 3(amino acids 62-72) of the amino acid sequence of ubiquitin as shown inSEQ ID NO: 1 and optionally, further comprising mutations in region 1(amino acids 2-14) of the amino acid sequence of ubiquitin as shown inSEQ ID NO: 1. The present disclosure also provides a nucleic acidencoding a ubiquitin enzyme inhibitor comprising an amino acid mutationin at least one of region 1 (amino acids 2-14), region 2 (amino acids42-49), or region 3 (amino acids 62-78) in the amino acid sequence of aubiquitin variant (Ubv) set forth in SEQ ID NO:21.

In another embodiment, there is provided a nucleic acid moleculeencoding a ubiquitin enzyme inhibitor comprising mutations in at leastone of positions 2, 4, 6, 8, 9, 10, 11, 12, 14, 35, 37, 39, 40, 42, 44,46, 47, 48, 49, 62, 63, 64, 66, 68, 70, 71 or 72 of the amino acidsequence of ubiquitin as shown in SEQ ID NO:1, optionally in at leastone of positions 2, 4, 6, 8, 9, 10, 11, 12, 14, 62, 63, 64, 66, 68, 70,71 or 72 of the amino acid sequence of ubiquitin as shown in SEQ ID NO:1or in at least one of positions 62, 63, 64, 66, 68, 70, 71 or 72 of theamino acid sequence of ubiquitin as shown in SEQ ID NO:1. In anotherembodiment, the ubiquitin enzyme inhibitor comprises mutations in atleast 2, 4, 6 or 8 of the positions.

In one embodiment, the disclosure provides a nucleic acid moleculeencoding a USP inhibitor comprising a mutation at position 68,optionally Tyr or Phe for His, of the amino acid sequence of ubiquitinas shown in SEQ ID NO: 1. In another embodiment, the disclosure providesa nucleic acid molecule encoding a USP inhibitor comprising a Val to Leumutation at position 70 of the amino acid sequence of ubiquitin as shownin SEQ ID NO: 1.

In a further embodiment, the disclosure provides a nucleic acid moleculecomprising the nucleic acid sequence as shown in SEQ ID NO: 13 orencoding a USP8 inhibitor comprising the sequence as shown in SEQ ID NO:2 (Ub variant 8.2) or a nucleic acid molecule comprising the nucleicacid sequence as shown in SEQ ID NO: 14 or encoding a USP8 inhibitorcomprising the sequence as shown in SEQ ID NO: 3 (Ub variant 8.3). Inanother embodiment, there is provided a nucleic acid molecule comprisinga nucleic acid sequence as shown in SEQ ID NO:15 or encoding a USP21inhibitor comprising the sequence shown in SEQ ID NO:4 (Ub variant21.1), a nucleic acid molecule comprising a nucleic acid sequence asshown in SEQ ID NO:16 or encoding a USP21 inhibitor comprising thesequence as shown in SEQ ID NO:5 (Ub variant 21.2), a nucleic acidmolecule comprising a nucleic acid sequence as shown in SEQ ID NO:17 orencoding a USP21 inhibitor comprising the sequence as shown in SEQ IDNO:6 (Ub variant 21.3), a nucleic acid molecule comprising a nucleicacid sequence as shown in SEQ ID NO:18 or encoding a USP21 inhibitorcomprising the sequence as shown in SEQ ID NO:7 (Ub variant 21.4) or anucleic acid molecule comprising a nucleic acid sequence as shown in SEQID NO:19 or encoding a USP21 inhibitor comprising the sequence as shownin SEQ ID NO:8 (Ub variant 21.5).

In another embodiment, the disclosure provides a nucleic acid encoding apolypeptide comprising at least one amino acid mutation at position 2,4, 6, 8, 9, 10, 11, 12, 14, 42, 44, 46, 47, 48, 49, 62, 63, 64, 68, 70,71, 72, 73, 74, 75, 76, 77, or 78 of the amino acid sequence of aubiquitin variant (Ubv) set forth in SEQ ID NO:21.

In addition, the present disclosure provides a nucleic acid whichencodes any of the polypeptides of SEQ ID NOS: 2-8 and 22-173.

In another embodiment, the disclosure provides a nucleic acid encodingany of the ubiquitin variants identified by the methods describedherein. A person skilled in the art will appreciate that the enzymeinhibitors, or more particularly, ubiquitin variants, of the disclosuremay be prepared in any of several ways, optionally, by recombinantmethods.

Accordingly, nucleic acid molecules encoding the enzyme inhibitors, ormore particularly, ubiquitin variants, may be incorporated in a knownmanner into an appropriate expression vector which ensures goodexpression of the proteins. Possible expression vectors include, but arenot limited to, cosmids, plasmids, or modified viruses (e.g. replicationdefective retroviruses, adenoviruses and adeno-associated viruses), solong as the vector is compatible with the host cell used. The expressionvectors may contain a nucleic acid molecule of the disclosure andregulatory sequences selected on the basis of the host cells to be usedfor expression, which is operatively linked to the nucleic acidmolecule. Operatively linked is intended to mean that the nucleic acidis linked to regulatory sequences in a manner which allows expression ofthe nucleic acid.

The disclosure therefore contemplates a recombinant expression vectorcomprising a nucleic acid molecule encoding an enzyme inhibitor, or moreparticularly, ubiquitin variant, as disclosed herein, and the necessaryregulatory sequences for the transcription and translation of theinserted protein-sequence.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes (Forexample, see the regulatory sequences described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). Selection of appropriate regulatory sequences isdependent on the host cell chosen, and may be readily accomplished byone of ordinary skill in the art. Examples of such regulatory sequencesinclude: a transcriptional promoter and enhancer or RNA polymerasebinding sequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other sequences, such as an origin of replication,additional DNA restriction sites, enhancers, and sequences conferringinducibility of transcription may be incorporated into the expressionvector.

The recombinant expression vectors may also contain a selectable markergene which facilitates the selection of host cells transformed ortransfected with a recombinant molecule of the disclosure. Examples ofselectable marker genes are genes encoding a protein such as G418 andhygromycin which confer resistance to certain drugs, β-galactosidase,chloramphenicol acetyltransferase, firefly luciferase, or animmunoglobulin or portion thereof such as the Fc portion of animmunoglobulin optionally IgG. Transcription of the selectable markergene is monitored by changes in the concentration of the selectablemarker protein such as β-galactosidase, chloramphenicolacetyltransferase, or firefly luciferase. If the selectable marker geneencodes a protein conferring antibiotic resistance such as neomycinresistance transformant cells can be selected with G418. Cells that haveincorporated the selectable marker gene will survive, while the othercells die. This makes it possible to visualize and assay for expressionof recombinant expression vectors. It will be appreciated thatselectable markers can be introduced on a separate vector from thenucleic acid of interest.

The recombinant expression vectors may also contain genes which encode afusion moiety which provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. For example, a proteolytic cleavage site may beadded to the target recombinant protein to allow separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Typical fusion expression vectors include pGEX(Amrad Corp., Melbourne, Australia), pMal (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the recombinant protein.

Recombinant expression vectors can be introduced into host cells toproduce a transformed host cell.

Suitable host cells include a wide variety of eukaryotic host cells andprokaryotic cells. For example, the enzyme inhibitors, or ubiquitinvariants, may be expressed in yeast cells or mammalian cells. Othersuitable host cells can be found in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1991). Inaddition, the enzyme inhibitors, or ubiquitin variants, may be expressedin prokaryotic cells, such as Escherichia coli (Zhang et al., Science303(5656): 371-3 (2004)). In addition, a Pseudomonas based expressionsystem such as Pseudomonas fluorescens can be used (US PatentApplication Publication No. US 2005/0186666, Schneider, Jane C et al.).

Accordingly, the disclosure provides a host cell comprising the nucleicacid sequences or recombinant expression vectors disclosed herein.

The nucleic acid molecules disclosed herein may also be chemicallysynthesized using standard techniques. Various methods of chemicallysynthesizing polydeoxynucleotides are known, including solid-phasesynthesis which, like peptide synthesis, has been fully automated incommercially available DNA synthesizers (See e.g., Itakura et al. U.S.Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; andItakura U.S. Pat. Nos. 4,401,796 and 4,373,071).

The enzyme inhibitors, or more particularly, ubiquitin variants, mayalso be prepared by chemical synthesis using techniques well known inthe chemistry of proteins such as solid phase synthesis (Merrifield, J.Am. Chem. Assoc. 85:2149-2154 (1964); Frische et al., J. Pept. Sci.2(4): 212-22 (1996)) or synthesis in homogenous solution (Houbenweyl,Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme,Stuttgart (1987)).

The disclosure also provides the various ubiquitin variants for use inscreening small molecule libraries for inhibitors of the ubiquitinpathway, including inhibitors of USPs, OTUs, E3 ligases andE2-conjugating enzymes.

The disclosure also provides the various ubiquitin variants describedherein for use in the treatment of a range of diseases, includingcancer. As used herein, the term “cancer” refers to any type of cancer,including, but not limited to, ovarian cancer, leukemia, lung cancer,colon cancer, CNS cancer, melanoma, renal cancer, prostate cancer,pancreatic cancer, breast cancer, and the like.

The above disclosure generally describes the present disclosure. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of thedisclosure. Changes in form and substitution of equivalents arecontemplated as circumstances might suggest or render expedient.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitation.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1—Design, Synthesis and Testing of Ubiquitin 8 and 21Variants

Results

Human Ub was displayed in a monovalent format on the surface of M13bacteriophage as a fusion to the C-terminal domain of the p3 minor coatprotein, using a phagemid system used previously for the display ofantibodies and other proteins [Sidhu, Lowman et al. 2000; Sidhu, Li etal. 2004; Li, Xi et al. 2009]. By inspection of structures of Ub incomplex with several USPs, 27 Ub residues were identified that makecontact with the USP surface, and this residue set was defined as theUSP-binding site. These 27 residues could be conveniently divided intothree groups, with each group containing residues that are closetogether in the primary sequence, as follows: region 1 (residues Q2, F4,K6, L8, T9, G10, K11, T12 and T14), region 2 (residues G35, P37, D39,Q40, R42, I44, A46, G47, K48 and Q49) and region 3 (residues Q62, K63,E64, T66, H68, V70, L71 and R72) (FIG. 1). To construct a library thatsimultaneously targets all 27 residues for randomization, a methodologythat has been applied previously for the construction of high qualityantibody libraries was used in which more than 30 residues in fourdistinct regions of primary sequence were successfully randomized[Fellouse and Sidhu 2007]. Three mutagenic oligonucleotides (eachcorresponding to one of the three regions) were synthesized andincorporated into the Ub-encoding gene in the phage display vector. A“soft” randomization strategy was utilized in which the mutagenicoligonucleotides were designed to encode approximately 75% of thewild-type (wt) amino acid and 25% of a mixture of the other amino acidsat each targeted position [Fellouse and Sidhu 2007]. In addition, thestrategy allowed for the recovery of the full wt sequence in eachregion, in the event that the mutagenic oligonucleotide was notincorporated (˜30% of the population). In this way, the library wasbiased in favour of the wt sequence but allowed for significantdiversity across the entire USP-binding surface, and it was reasonedthat this would enable the selection of variants with mutations thatimprove affinity for a particular USP without drastically altering thebinding site.

A phage pool representing the Ub library was cycled through five roundsof selection for binding to immobilized USP8 or USP21. After the fifthround, 24 clones from each selection were tested for binding activity byphage enzyme-linked immunosorbent assay (ELISA) [Tonikian, Zhang et al.2007]. Clones that bound to the relevant USP, but not to a negativecontrol protein, were subjected to DNA sequencing, and this analysisrevealed two or five unique Ub variants that bound to USP8 or USP21,respectively (FIG. 2A). The binding profiles of these variants, and thatof wt Ub (Ub.wt (SEQ ID NO: 1)), were analyzed in further detail byphage ELISA against a panel of five USPs (USP8, USP21, USP2, USP7 andUSP10). As expected, each of the seven Ub variants recognized itscognate USP, and importantly, none of the clones exhibited detectablebinding to any of the four non-cognate USPs (FIG. 2B). Ub.wt (SEQ IDNO: 1) bound weakly to USP21 but did not exhibit detectable binding tothe other four USPs, consistent with low affinity binding that is belowthe detection limit of the monovalent phage display format.

Sequence analysis revealed both common and unique features amongst theclones selected for binding to the two USPs. In all cases, region 2 wascompletely conserved as the wt, suggesting that mutations in this regiondo not generally enhance affinity. Four of the five USP21-binding clonescontained mutations only in region 3, and one clone contained mutationsin regions 1 and 3. The two USP8-binding clones contain mutations inboth regions 1 and 3. Interestingly, all seven clones share a common Valto Leu mutation at position 70 and they all contain a mutation atposition 68, although the nature of the mutation differs slightlyamongst clones selected against the different targets (Tyr or Phesubstitute for His in clones that bind to USP8 or USP21, respectively).Aside from these common features, the binders for USP8 and USP21 differin sequence at other positions, and these differences likely conferspecificity. The Ub variants were cloned into a bacterial expressionvector and were purified from the soluble cytoplasmic fraction asHis-tag fusions. All variants were purified in good yield (˜10 mg perliter of culture) and were stable for months at 4° C. To test whetherthe Ub variants act as inhibitors of USP proteolytic activity, in vitroassays were used with the substrate Ub-AMC, which is prepared byC-terminal derivatisation of Ub with 7-amino-4-methylcoumarin (AMC).Active USPs cleave the Ub-AMC substrate and release fluorescent AMC.This is a well-established assay for probing USP activity and is alsoused to determine the affinity of Ub for USPs [Case and Stein 2006;Renatus, Parrado et al. 2006].

These assays showed that the activity of USP8 was inhibited by Ubv.8.2(SEQ ID NO: 2) but not by either Ub.wt (SEQ ID NO: 1) or Ubv.21.3 (SEQID NO: 6). Conversely, the activity of USP21 was inhibited by Ubv.21.3(SEQ ID NO: 6) but not by either Ub.wt (SEQ ID NO: 1) or Ubv.8.2 (SEQ IDNO: 2) (FIG. 3A). Full dose-response curves revealed that Ubv.8.2 (SEQID NO: 2) is an extremely potent inhibitor of USP8 activity (IC₅₀=6 nM)and Ubv.21.3 (SEQ ID NO: 6) is a potent inhibitor of USP21 activity(IC₅₀=40 nM) (FIG. 3B).

The structures of USP21 and USP8 were determined in complex with thecorresponding inhibitor to confirm the details of the inhibition (FIG.4). Overall, the USP and the Ub fold were found to be preserved and tobe similar to other USP Ub complex structures. For USP21, the resultingstructure was compared with the USP21-Ub complex structure (pdb entry:3I3T). Interestingly, the C-terminus of the Ub-variant in theUSP21-Ubv.21.4 (SEQ ID NO: 7) crystal structure appears to be cleavedalthough the Ubv.21.4 (SEQ ID NO: 7) was purified with additionalC-terminal residues (FIG. 4A). This shows that the protein is capturedin an active conformation. Additionally, to stabilize the crystallizedcomplex of USP21-Ub, the Ub is covalently attached to the active sitecysteine of USP21. In the USP21-Ubv.21.4 (SEQ ID NO: 7) structure, thisis not necessary since the variant binds with a high affinity to thebinding site to form a stable complex at room temperature. In general,it was observed that former repulsive interactions necessary for correctproduct release have been replaced with attractive interactions. Forexample, the mutation E64W in the ligand replaces the repulsiveinteraction with D438 on USP21. At the same time, the mutation H68Freplaces a water-mediated hydrogen bond with a hydrophobic interaction.Unexpectedly, this mutation is conserved amongst the selected bindershowever does not necessarily produce the same environment since it playsa different role in the USP8:Ubv.8.2 (SEQ ID NO: 2) structure. Finally,the more conservative mutation V70L improves hydrophobic contactsbetween the inhibitory Ubv.21.4 (SEQ ID NO: 7) and USP21 compared toUb.wt (SEQ ID NO: 1).

In the USP8:Ubv.8.2 (SEQ ID NO: 2) structure the C-terminus does notalign with the active site of the protein (FIG. 4B). Although notvisible in the crystal structure, the extended C-terminus is stillpresent as shown by mass-spec of the crystallized sample. In contrast toUbv.21.4 (SEQ ID NO: 7), Ubv.8.2 (SEQ ID NO: 2) contains 12 mutationsacross region 1 and region 3 of the interaction interface of Ub. Thecatalytically non-active binding mode is explained by a slight twist ofthe interactions of the C-terminal β-strand, where instead ofinteracting with the lower parts of the zinc-finger region it interactswith the α-helix of the palm-domain of the USP flanking the bindingpocket. This rotation is mainly caused by two hydrogen bonds formedbetween mutations in region 1 (Ubv.8.2 (SEQ ID NO: 2):Q2R and USP8:E895and Ubv.8.2 (SEQ ID NO: 2):K11R and USP8:E947 respectively) and isfurther stabilized by an additional hydrogen bond formed between Ubv.8.2(SEQ ID NO: 2):H68Y and USP8:D878.

Materials and Methods

Construction of the Ub-Library

The Ub library was constructed based on the phagemid pLB0131.F. Thisplasmid encodes under the control of an IPTG inducible lac-promoter foran N-terminal fusion protein of the DsbA signal sequence, the Flag-tag(DYKDDDK (SEQ ID NO:9), Ub and a truncated version of the minor coatprotein p3 of filamentous phage M13. Single stranded DNA template ofthis plasmid was prepared using previously described methods [Tonikian,Zhang et al. 2007]. The oligonucleotides targeting region 1 (poll),region 2 (pdl2) and region 3 (pdl3) were designed as follows: pdl1 (SEQID NO:10): GAC GAT GAC AAA ATG (N2)(N1)(N3) ATT (N4)(N4)(N2) GTG(N1)(N1)(N1) ACC (N2)(N4)(N4) (N1)(N2)(N3) (N3)(N3)(N3) (N1)(N1)(N3)(N1)(N2)(N2) ATC (N1)(N2)(N2) CTC GAG GTT GAA CCC, pdl2 (SEQ ID NO:11):ATC CAG GAT AAG GAA (N3)(N3)(N1) ATT (N2)(N2)(N4) CCT (N3)(N1)(N4)(N2)(N1)(N3) CAG (N1)(N3)(N1) CTG (N1)(N4)(N2) TTT (N3)(N2)(N4)(N3)(N3)(N2) (N1)(N1)(N3) (N2)(N1)(N3) CTG GAA GAT GGA CGT, pdl3 (SEQ IDNO:12): TCT GAC TAC AAT ATT (N2)(N1)(N1) (N1)(N1)(N3) (N3)(N1)(N3) TCT(N1)(N2)(N4) CTT (N2)(N1)(N4) CTT (N3)(N4)(N3) (N4)(N4)(N3) (N1)(N3)(N1)CTT CGT GGT GGT GGC. For each oligonucleotide the place holder N1describes a mixture of nucleotides A 73% C 9% G 9% and T 9%; N2describes a mixture of A 9%, C 73%, G 9% and T 9%; N3 describes amixture of A 9% C 9%, G 73% and T 9%; N4 describes a mixture of A 9%, C9%, G 9% and T 73%. Different mutational loads are conceivable dependenton the purpose of the application. All three oligonucleotides were usedsimultaneously in the mutagenesis using previously established methods[Tonikian, Zhang et al. 2007]. In total a library diversity of 7.5 10¹⁰unique Ub-variants was achieved. Phage pools representing the naïvepeptide library were produced from E. coli SS320 cultures grownovernight at 37° C. in superbroth media supplemented with 25 μg/mlkanamycin, 100 μg/ml carbenicilin, 0.4 mM IPTG. Phage were harvested byprecipitation with 20% PEG-8000/2.5M NaCl and resuspended at a finalconcentration of 10¹³ phage/mL in assay buffer (PBS, 0.5% BSA and 0.5%Tween 20).

Selection of Inhibitors

The resulting library was independently selected against the catalyticdomains of USP21 (Uniprot accession number Q9UK80: residues 209-564) andUSP8 (Uniprot accession number P40818: residues 764 to 1118). Both USPswere biotinylated using commercially available N-hydroxysuccinimidyld-biotin-15-amido-4,7,10,13-tetraoxapentadecylate (NHS-PEO₄-Biotin)following the manufacturer's instructions (Thermo Fisher Scientific,Rockford, II 61105 USA). 100 μl Neutravidin (50 μg/ml in PBS) (ThermoFisher Scientific, Rockford, II 61105, USA) was coated overnight at 4°C. on a Maxisorp micro titer plate (Nalge Nunc International, Rochester,N.Y., USA) (100 μL per well). The wells were blocked for 2 hours withblocking buffer (PBS, 0.2% BSA, 0.5% Tween 20). The biotinylated targetproteins USP8 and USP21 were diluted into PBS to a final concentrationof 50 μg/ml and 100 μL were added to the micro titer plate. 100 μL ofubiquitin-displaying phage were added to each well and incubated for 2 hat 4° C. Non-binding phage were removed by washing the micro-titer plate8 times. Bound phage were eluted for 15 min with 100 μL 0.1 N HCl andimmediately neutralized by addition of 1 M Tris. E. coli XI1 blue wasgrown to an OD₆₀₀ of 0.6 and an aliquot of this culture was infectedwith the eluted phage in a ratio of 1:10. After incubation for 30 min at37° C. while shaking at 200 rotations per minute (rpm) helper phageM13KO7 was added to a multiplicity of infection (MOI) of 10 andincubated for 1 h at 37° C. while shaking at 200 rpm. This culture wastransferred to 30 ml of freshly prepared 2YT containing 25 μg/mlkanamycin, 100 μg/ml carbenicilin, 0.4 mM IPTG and incubated over nightat 37° C. while shaking at 200 rpm. From this culture of XL1 blue thephage for the second round of selection were prepared by precipitationwith 20% PEG-8000/2.5 M NaCl. Phage were resuspended in 1 ml of assaybuffer and used in a 2 second round of selection. All rounds on wardswere done at room temperature and washing steps after phage incubationwere increased from 10 in the 2^(nd) round to 16 in the 5^(th) round ofselection. After the 5^(th) round, individual ub variants wereidentified by single clone ELISA using established techniques [Pearce,Potts et al. 1997]. By sequencing of the encoding DNA 5 Ub-variants withbinding activity to USP21 and 2 Ub-variants with binding activity toUSP8 were identified (FIG. 2A).

Specificity Test

The specificity of the selected binders was tested against a set ofunrelated USPs in phage ELISA format (Pearce, 1997 #533). As test setthe biotinylated catalytic domains of USP2 (Uniprot entry Q75604:residues 262-605), USP7 (Uniprot entry Q93009: residues 207-533) andUSP10 (Uniprot entry Q14694: residues 385-798) were used. USP8 and USP21were included as positive control for the corresponding Ub-variants.From individual colonies encoding the variants Ubv.21.1 (SEQ ID NO: 4),Ubv.21.2 (SEQ ID NO: 5), Ubv.21.3 (SEQ ID NO: 6), Ubv.21.4 (SEQ ID NO:7), Ubv.21.5 (SEQ ID NO: 8), Ubv.8.1, Ubv.8.2 (SEQ ID NO: 2) and Ub.wt(SEQ ID NO: 1) 450 μl of 2YT containing 25 μg/ml kanamycin, 100 μg/mlcarbenicilin, 0.4 mM IPTG and 10¹⁰ cfu/ml helper phage M13KO7 wereinoculated and incubated over night at 37° C. while shaking at 200 rpm.Cultures were centrifuged at 10000 g for 10 min at 4° C. and thesupernatant was diluted 1:10 in PBS and directly added to theimmobilized USP. The USPs were immobilized on a micro-titer plate asdescribed above. After incubation of the diluted phage containingculture supernatant for 1 h, the ELISA plate was washed 3 times withPBST 0.1 and 100 μl 1:5000 dilution of commercially available anti-M13antibody horseradish peroxidase (Amersham-Pharmacia, Piscataway, N.J.)was added and incubated for 1 h. After a second wash, the ELISA wasdeveloped with 100 μl TMB peroxidase substrate (KPL, Gaithersburg, Md.,USA). The reaction was stopped after 5 min by addition of 100 μl 1 MH₃PO₄ and the signal was recorded at 450 nm in an ELISA reader (FIG. 2B).

Characterization of USP21 Inhibition

The activity of USP21 at a concentration of 25 nM in HEPES-buffer (50 mMHEPES, pH 7.5., 0.01% Tween 20 and 10 mM DTT) was measured with Ub-AMCsubstrate at 1.0 μM in HEPES-buffer at RT. The release of7-amino-4-methycoumarine was measured at 460 nm in fluorescencespectrometer at an excitation wavelength of 380 nm. The 1050 value wasdetermined as the concentration of Ub variant that reduces USP activityby 50% as normalized to the activity in the absence of Ub variants. The1050 was determined by incubating the enzyme USP21 with the Ub variantswith binding activity to USP21 at concentrations 1 μM, 0.5 μM, 125 nM,100 nM, 75 nM, 50 nM, 25 nM, 10 nM and 6 nM. The variants Ubv.21.3 (SEQID NO: 6) and Ubv.21.4 (SEQ ID NO: 7) were found to inhibit the USP21with an 1050 of 40 nM and 70 nM, respectively.

Usp21 solution (500 μl, 2.2 mg/ml in 20 mM Tris-HCl, pH 7.0, 0.5 M NaCl,5% glycerol and 2 mM dithiothreitol) was mixed with Ubv.21.4 (SEQ ID NO:7) solution (100 μl, 4.9 mg/ml), which resulted in Usp21:Ubv.21.4 (SEQID NO: 7) molar ratio of 1:1.6. The mixture was incubated for 1 h atambient temperature (294 K) followed by incubation for 16 h at 281 K andconcentrated by ultrafiltration to a final volume of 150 μl, whichresulted in Usp21 concentration of 9 mg/ml. Crystals of theUsp21-inhibitor complex were grown at 291 K using the hanging dropmethod by mixing equal volumes of the above complex solution andCrystallization Buffer (11% polyethyleneglycol 4000, 0.1 M sodiumcitrate, pH 5.3, 0.1 M ammonium acetate and 0.5 mM TCEP). The crystalswere cryoprotected by immersion in the Crystallization Buffersupplemented with 25% (v/v) glycerol and placed in liquid nitrogen.

Diffraction data from a crystal of the Usp21 catalytic domain in complexwith Ubv.21.4 (SEQ ID NO: 7) inhibitor was collected on a Rigaku FR-ESuperbright generator equipped with an R-AXIS IV++ detector. The dataset was integrated and scaled using the HKL2000 program suite. Thestructure was solved by molecular replacement techniques using theprogram PHASER and search model PDB entry 3I3T. Iterative model buildingusing the graphics program Coot and refinement package REFMAC5 led to amodel with an R factor of 21.77 (Rfree 27.32%) for data between 20-2.7Å.

Characterization of USP8 Inhibition

The activity of USP8 at a concentration of 1 nM in HEPES-buffer (50 mMHEPES, pH 7.5., 0.01% Tween 20 and 10 mM DTT) was measured with Ub-AMCsubstrate at 1.0 μM in HEPES-buffer at RT. The release of7-amino-4-methycoumarine was measured at 460 nm in fluorescencespectrometer at an excitation wavelength of 380 nm. The 1050 value wasdetermined as the concentration of Ub variant that reduced USP activityby 50% and is normalized to the activity in the absence of Ub variants.The 1050 was determined by incubating the enzyme USP8 with the Ubvariants with binding activity to USP8 at concentrations 100 nM, 75 nM,50 nM, 25 nM, 10 nM, 5 nM 2.5 nM, 1 nM, 0.8 nM and 0.5 nM. The variantsUbv.8.3 (SEQ ID NO: 3) was found to inhibit USP8 with an 1050 of 6 nM.

Usp8 solution (50 μl, 7.6 mg/ml in 20 mM Tris-HCl, pH 7.0, 0.5 M NaCl,5% glycerol and 2 mM dithiothreitol) was mixed with Ubv.8.2 (SEQ ID NO:2) (30 μl, 3.5 mg/ml), which resulted in Usp8: inhibitor molar ratio of1:1. Before setting crystallization plate, the mixture was incubated for1 h at ambient temperature (294 K) followed by incubation for 16 h at281 K. Crystals of the Usp8-inhibitor complex were grown at 291 K usingthe hanging drop method by mixing equal volumes of the above complexsolution and Crystallization Buffer (24% polyethyleneglycol 3350, 0.1 Mbis-Tris, pH 6.0, 0.2 M ammonium acetate and 0.5 mM dithiothreitol). Thecrystals were cryoprotected by immersion in the Crystallization Buffermixed (1:1, v/v) with cryoprotecting mixture that consisted of 20% (w/v)sucrose, 4% (w/v) glucose, 18% (v/v) glycerol and 18% (v/v) ethyleneglycol in water and placed in liquid nitrogen.

Diffraction data from a crystal of the Usp8 catalytic domain in complexwith Ubv.8.2 (SEQ ID NO: 2) inhibitor was collected on a MAR-300detector at the Canadian Light Source beamline CMCF 08ID-1. The data setwas integrated and scaled using the HKL2000 program suite. The structurewas solved by molecular replacement techniques using the program PHASERand search model PDB entry 2GFO and 3MTN. Iterative model building usingthe graphics program Coot and refinement package REFMAC5 led to a modelwith an R factor of 17.8 (Rfree 24.2%) for data between 35-2.6 Å.Initial parameters for TLS refinement were obtained from the TLSMD webserver.

Example 2—New Ubiquitin Library Design

Ubiquitin variants, previously selected against USP2, USP5, USP7, USP10and USP46, had heavily altered region 2 sequences which led to misfoldednonspecifically binding clones. Accordingly, the diversity in region 2was restricted to avoid misfolding and, therefore, the currentlydesigned ubiquitin variant proteins comprises only 6 amino acid residuescompared to the original 10 residues in region 2.

Additionally, the randomized region beyond the C-terminus of ubiquitinwas extended to target directly the active site of USPs. TheUSP21:Ubv21.4 inhibitor structure (pdb code: 3MTN) showed that theC-terminus (residues 73 through residues 76) of ubiquitin variant 21.4is located in the active site of the USP protein. Therefore, a ubiquitinvariant was extended by two additional residues to derive an inhibitoryvariant which spans the active site of the USPs, which could result inoptimized contacts in the active site and lead to a more efficientinhibition of the USPs. Additionally, ubiquitin variants withmodifications at the C-terminus could reveal critical intramolecularcontacts which could facilitate the development of small molecule (<1000Da) inhibitors of this enzyme class.

Variant design is set out as follows:

Region 1: Q2, F4, K6, L8, T9, G10, K11, T12, T14

Region 2: R42, 144, A46, G47, K48, Q49

Region 3: Q62, K63, E64, H68, V70, L71, R72, L73, R74, G75, G76, G76a(or G77), G76b (orG78)

Example 3—New Target Proteins

A newly designed phage-displayed library, as set out in Example 2 above,was used in selection experiments against USP2a, USP5, USP10 and USP48.At the same time, binders against the E3-ligases Nedd4, ITCH and linearubiquitin binding motifs (UIM) were selected. USP8 and USP21 wereincluded as positive controls for the selection.

Binders to USP2a

USP2a acts in cooperation with USP7/HAUSP as a positive regulator ofHDM2, the major ubiquitin E3 ligase which directs p53 for proteasomaldegradation (Priolo, C. 2006). Overexpression of USP2a innon-transformed cells results in an oncogenic phenotype and was shown toprevent apoptosis through chemotherapeutic drugs. Additionally,RNAi-mediated silencing of USP2a leads to an increased apoptosis ofseveral prostate cancer cell lines, which makes USP2a a therapeuticallyattractive target in prostate cancer.

The catalytically active domain of USP2a (residues 251-605) wasexpressed, biotinylated and immobilized for selection on aneutravidin-coated surface. After 5 consecutive rounds of phage display,individual clones were analyzed for binding activity (FIG. 5B). Intotal, 26 variants (SEQ ID NOS: 26-47) with specificity for USP2a (FIG.5A) were identified. Using a competitive ELISA, 6 lead candidates withan affinity of 25 nM or better were identified.

Binders to USP5

USP5/IsoT processes unanchored poly-Ub chains and plays an importantrole in Ub recycling. Knockdown of USP5 expression by shRNA leads toincreased free poly-Ub and increased transcription of the p53 gene(Dayal, S. et al. 2009). Residues 1-835 of USP5 were expressed in thesame format as USP2a, as described herein above. After 5 rounds ofselection, 21 unique USP5-binding Ubvs (SEQ ID NOS: 48-68) wereidentified (FIG. 6A). All 21 selected Ubvs have very good specificityfor USP5 (FIG. 6B).

Binders to USP10

USP10 is a cytoplasmic protease which deubiquitinates ubiquitin-labeledp53 and contributes to its stabilization. During DNA damage response,USP10 is stabilized and is involved in nuclear activation of p53 (Yuan,J. 2010). Residues 371-798 of USP10, which comprise the catalytic domainof the enzyme, were expressed. Surprisingly, only one unique ubiquitinvariant, Ubv.10.1 (SEQ ID NO: 69), was identified after 5 rounds ofselection (FIG. 7A). Ubv.10.1 (SEQ ID NO: 69) has 17 mutationsdistributed evenly throughout the reading frame of Ub (FIG. 7A).Ubv.10.1 (SEQ ID NO: 69), showed high specificity for USP10 and nobinding activity to other USPs (FIG. 7B).

Binders to USP48

Using genome-wide RNA interference screens, it was found that USP48 isessential for the viability of pancreatic cancer cells that aredependent on the mutant KRAS Small hairpin RNA (shRNA)-mediatedknockdown of USP48 shows that certain pancreatic cancer cell linesrequire USP48 to survive. Residues 38-478 of USP48 were expressed withan N-terminal HIS-tag and a C-terminal in vivo biotinylation tag(AVI-tag). After 5 rounds of selection, 5 unique ubiquitin variants (SEQID NOS: 70-74) that showed high binding specificity for USP48 wereidentified (FIGS. 8A and 8B).

NEDD4 Binders

Neural precursor cell expressed developmentally down-regulated protein 4(Nedd4) is an E3 ligase which accepts ubiquitin from an E2Ub-conjugating enzyme and transfers it to other proteins. Nedd4functions as a positive regulator of dendrite development andubiquitinates Rap2a, a member of the Ras oncogene family (Kawabe et al.,2010). Nedd4 ubiquitinates multiple receptor tyrosine kinases, such asEGF receptors, FGF receptor, and IGF1 receptor and functions in theirendocytosis and degradation. Additionally, Nedd4 has been identified asa regulator of the tumor suppressor PTEN and is found to be upregulatedin bladder and prostate carcinomas. Nedd4 is composed of several WWdomains and a catalytically active HECT domain. The WW domain is ahighly conserved protein interaction module comprising 35-40 aminoacids. The HECT domain (Homologous to the E6-AP Carboxyl Terminus) is anapproximately 40 kDa (350-amino acid) catalytic domain found at thecarboxyl terminus of HECT-class E3 ubiquitin protein ligases. Nedd4 wasincluded to test additional proteins involved in ubiquitination. Highlyspecific binders to the HECT-domain of Nedd4 (residues 510-900) wereselected. In total, 24 unique ubiquitin variants which bind Nedd4 (SEQID NOS: 75-98), and which had mutations in all regions of ub, wereidentified (FIGS. 9A and 9B). Analysis showed that the mutations G10R,I44F, Q49K, H68Y, L71 K and G76M are conserved. These mutations couldmake beneficial contacts to residues in Nedd4. For example, G10R isimplicated for its role in forming a new salt bridge to D614 in Nedd4and, thereby, increasing affinity.

ITCH Binders

The ubiquitin ligase, ITCH, acts as an E3 ubiquitin protein ligase. ITCHaccepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form ofa thioester and then directly transfers the ubiquitin to targetedsubstrates. ITCH catalyzes ‘Lys-29’-, ‘Lys-48’- and ‘Lys-63’-linkedubiquitin conjugation. ITCH belongs to the family of HECT-domain E3ligases and is closely related to Nedd4. Residues 483-862 of ITCH, whichcomprise the HECT domain, were expressed. After 5 rounds of selection, 6unique binders to ITCH (SEQ ID NOS: 99-104) (FIGS. 10A and 10B) wereidentified. The selected binders had an average of 16 mutationsdistributed over the Ub scaffold.

UIM Binders

Ubiquitin interaction motifs (UIM) are linear sequence motifs which arepart of other proteins. UIMs have been shown to bind ubiquitin and toserve as a specific targeting signal important for monoubiquitination.UIMs are often found in a variety of proteins involved in ubiquitinationand ubiquitin metabolism, or interact with ubiquitin-like modifiers. Forexample, UIMs play a major role in endosomal sorting and recognizeeither poly-ubiquitinated or mono-ubiquitinated substrates.

Selection for UIM binders was made against a linear peptidecorresponding to UIM1 (RPGM-1) of vacuole-protein-sorting 27 protein(VPS27p) of yeast. UIM1 is part of a yeast protein, VPS27, and isresponsible for the recognition of ubiquitin in ubiquitinated proteins.UIM1 is part of the endosomal sorting complex ESCRT0 which isresponsible for the trafficking and degradation of ubiquitinatedreceptors, such as EGFR. An engineered variant, therefore, can competewith Ub.wt (SEQ ID NO: 1) binding and interfere with endosomal sorting.

Two peptides, UIM1a (RPGM-2) and IUM1b (RPGM-3), comprising pointmutations of the UIM1 abolishing UB binding were identified.

(SEQ ID NO: 170) UIM1: GGGGAADEEELIRKAIELSLKESRNSGGY (SEQ ID NO: 171)UIM1a: GGGGAADEEELIRKAIELALKESRNSGGY (SEQ ID NO: 172) UIM1b:GGGGAADEEELIRKLIELSLKESRNSGGY

Similar to Nedd4, these peptide motifs were used in selections to expandon other intracellular ub binding moieties. High-affinity bindersagainst these motifs could be used to interfere with endosomal sortingmechanisms and are considered valuable research tools. Four uniquebinders recognizing UIM1; 6 unique binders in the UIM1a selection; and15 unique binders in the UIM1b selection were identified (SEQ ID NOS:105-129) (FIG. 11A). All Ubvs showed no binding to other ubiquitinbinding moieties, such as USPs or E3 ligases. However, the selectedvariants had varying degrees of specificity amongst themselves. Forexample, several variants recognizing all three UIM1 peptides (UIM1,UIM1a, and UIM1b), two UIM1 peptides, or one UIM1 peptide, respectively,were identified (FIGS. 11B-D).

Binders for USP8 and USP21

Additional experiments for USP8 and USP21 binders were carried out withthe 78-amino acid Ub.wt variant of SEQ ID NO: 21. As positive controls,all selection experiments for USP8 and USP21 were included. Inhibitorsfor both enzymes, USP8 and USP21, were previously developed as describedherein above in Example 1. In these new experiments, several more uniquebinders against USP8 (11 ubv, SEQ ID NOS: 130-140) and USP21 (29 ubv,SEQ ID NOS: 141-169) were identified. These 40 new Ubvs showed highspecificity (FIGS. 12 and 13), comparable to specificity of USP8 andUSP21 binders previously identified.

Example 4—Measurement of Ubv.21.4 (SEQ ID NO: 7) and Ubv.8.2 (SEQ ID NO:2) IC50

Inhibition experiments with Ubv.8.2 (SEQ ID NO: 2) and Ubv.21.4 (SEQ IDNO: 7) were repeated with some modifications. Previous measurementsshowed a slight salt effect on proteolytic activity, which led to anunderestimation of the affinity of Ubv.21.4 (SEQ ID NO: 7). Therefore,C-terminally truncated versions of two variants, Ubv.8.2 (SEQ ID NO: 2)and Ubv.21.4 (SEQ ID NO: 7), were measured, thereby avoiding activationand conjugation to other intracellular proteins. FIGS. 14A and B showthe new dose-response curves.

Example 5—Intracellular Inhibition of USP21 by Co-Expression of Ubv.21.4

Previous experiments already have established the inhibitory potentialof Ubv.8.2 (SEQ ID NO: 2) and Ubv.21.4 (SEQ ID NO: 7) on USP8 and USP21,respectively, in vitro. Both binders inhibit their cognate USP with asingle digit nanomolar IC50. Thus, experiments were carried out todetermine whether Ubv.21.4 (SEQ ID NO: 7) inhibits USP21 activity invivo. USP21 inhibits TNFα and RIP1-induced NFκB activation in adose-dependent manner (FIGS. 15A and B) (Xu, G. F. et al. 2010).

When increasing amounts of either Ub.wt (SEQ ID NO: 1) or Ubv.21.4 (SEQID NO: 7) were co-expressed with USP21, Ubv.21.4 (SEQ ID NO: 7), but notUb.wt (SEQ ID NO: 1), suppressed the inhibitory effect of USP21 on TNFα-and RIP1-induced NF-κB activation (FIGS. 15C and D). Consequently,Ubv.21.4 (SEQ ID NO: 7), but not Ub.wt (SEQ ID NO: 1), rescued theinhibitory effect of USP21 on RIP1 polyubiquitination (FIG. 15E).Consistent with these results, USP21 co-immunoprecipitated with Ubv.21.4(SEQ ID NO: 7), but did not co-immunoprecipitate with Ub-wt, in 293Tcells co-transfected with plasmids to express these proteins (FIG. 15F).Taken together, these data show that Ubv.21.4 (SEQ ID NO: 7)specifically blocks USP21 activity through physical binding with USP21in the cells.

Experiments showing that Ubv.8.2 (SEQ ID NO: 2) inhibits USP8intracellularly are ongoing. It has been established, however, by massspectroscopy that both Ubv.8.2 (SEQ ID NO: 2) and Ubv.21.4 (SEQ ID NO:7) bind to their cognate USP in cells.

Example 6—Ubiquitin Inhibitors Cause Apoptosis in Cancer Cells and/orCause Tumor Regression

Lentiviruses can stably infect most cell types, including primary andnon-dividing cells, and lentiviral-based libraries can be used forgenetic selection screens (Moffat et al, 2006). Adapting Ub variantsinto lentiviruses accelerates discovery of inhibitors that induce aparticular phenotype, such as apoptosis and cell growth arrest, inrelevant cell types. Additionally, lentivirus allows direct in vivoexperiments in mouse models of disease. Thus, lentiviral-basedconstructs for stable and inducible expression of GFP-Ubv.8.2 (SEQ IDNO: 2) and GFP-Ubv.8.3 (SEQ ID NO: 3) are constructed. These ubiquitinvariants are used with USP8 inhibitors to explore effects on EGFRsignaling. The GFP-Ubv.8.2 (SEQ ID NO: 2) and GFP-Ubv.8.3 (SEQ ID NO: 3)constructs are used to generate EGFR-dependent cell lines harbouringthese Ub variants under the control of a constitutive ordoxycycline-inducible promoter. These experiments will be used tooptimize lentiviral delivery of ubiquitin variants. Multiple cancer celllines are screened in a high throughput manner to explore the phenotypiceffects that the ubiquitin inhibitors have on these cells.

Example 7—Ubiquitin Inhibitors for Screening Small Molecule Libraries

The ubiquitin variants are also used to screen small molecule compoundlibraries for the inhibition of USPs, OTUs, E3 ligases andE2-conjugating enzymes. In such a screen, a high affinity ubiquitinvariant is labeled with a fluorescent dye allowing the measurement offluorescence polarization (FP) of an enzyme ubiquitin variant complexand the free ubiquitin variant (Hafner et al., 2008). FP directlycorrelates with the hydrodynamic radius and diffusion of labeledproteins in solution, i.e., a labeled ubiquitin variant dissociated froman enzyme complex has lower FP values compared to the complex boundform. Therefore, a fluorescence-labeled high affinity ubiquitin variantcould be displaced from the active site of an enzyme by a small moleculewhich results in a decrease of FP compared to the intact complex.Additionally, the affinity of a small molecule binding to the activesite directly correlates with the affinity of the displaced engineeredubiquitin variant. These screens are performed in a high-throughputfashion and could potentially result in a series of small moleculesspecifically inhibiting enzymes of the ubiquitination pathways in vitroand in vivo.

While the present disclosure has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the disclosure is not limited to the disclosed examples.To the contrary, the disclosure is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

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The invention claimed is:
 1. A specific inhibitor of a deubiquitinatingenzyme, wherein the deubiquitinating enzyme is USP8, and wherein theinhibitor is ubiquitin variant comprising the amino acid sequence setforth in SEQ ID NO: 2 or
 3. 2. The inhibitor of claim 1, wherein theubiquitin variant comprises the amino acid sequence set forth in SEQ IDNO:
 2. 3. The inhibitor in claim 1, wherein the ubiquitin variantcomprises the amino acid sequence set forth in SEQ ID NO:
 3. 4. Anucleic acid encoding the inhibitor of claim
 1. 5. The nucleic acid ofclaim 4 in a recombinant expression vector.
 6. The nucleic acid of claim5 in a host cell.
 7. The nucleic acid of claim 4, wherein the ubiquitinvariant comprises the amino acid sequence set forth in SEQ ID NO:
 2. 8.The nucleic acid of claim 4, wherein the ubiquitin variant comprises theamino acid sequence set forth in SEQ ID NO: 3.